Prevalence, genetic diversity, environmental shedding and associated factors of Giardia duodenalis in cattle (Bos taurus) and canids (Canidae) in Latvia = Giardia duodenalis izplatība, ģenētiskā daudzveidība, vides piesārņojums un to ietekmējošie faktori govīm (Bos taurus) un suņu dzimtas (Canidae) dzīvniekiem Latvijā

The doctoral thesis “Prevalence, genetic diversity, environmental shedding and associated factors of Giardia duodenalis in cattle (Bos taurus) and canids (Canidae) in Latvia” by Maira Mateusa was conducted between 2019 and 2025 at the Institute of Food and Environmental Hygiene of the Faculty of Veterinary Medicine and at the Institute of Food Safety, Animal Health and Environment “BIOR”, the Laboratory of Microbiology and Pathology, Parasitology and Microbial Genomic groups. The study was done in three study periods. The first study period was focused on the prevalence, cyst load, genetic diversity, risk and protective factors associated with G. duodenalis prevalence in cattle in Latvia. The second study period covered the prevalence, cyst load, and genetic diversity of G. duodenalis in domestic dogs, and risk analysis was performed to identify factors that could influence the infection in domestic dogs. The third study period was focused on the prevalence, cyst load intensity, and genetic diversity of G. duodenalis in red foxes and raccoon dogs. Additionally, statistical analysis was performed to estimate the contribution of studied animal species to the contamination of the general environment with G. duodenalis cysts with the focus on zoonotic assemblage A. The first study period was from March 2019 to March 2021. A total of 973 cattle fecal samples from 32 herds were collected. Feces were examined using immunofluorescence staining followed by microscopic examination. Microscopically positive samples were subjected to G. duodenalis assemblage differentiation targeting the beta-giardin gene with restriction length fragment polymorphism analysis (RFLP). A questionnaire was developed to gather data on herd management practices and to analyze potential factors affecting the prevalence of G. duodenalis in studied herds. Questionnaires were filled out by interviewing the herd owners or responsible veterinarians. Generalized linear mixed modeling (GLMM) fit by maximum likelihood (Laplace approximation) was performed to identify risk and protective factors associated with the prevalence of G. duodenalis. All factors were assessed at the herd level, excluding animal age, sex, breed, and diarrhea (present/absent). Age was expected to be an important effect-modifying variable; therefore, data on age in days were included in risk and protective factor calculation models. The second study period lasted from April 2020 until May 2022. A total of 373 dog feces were tested. The same methodology as for cattle was applied for G. duodenalis cyst and assemblage detection. The dog owners filled out a questionnaire. The generalized linear model (GLM) of the binomial family was performed to identify risk and protective factors associated with G. duodenalis infection. The third study period lasted from February 2020 to January 2025, where feces from red foxes and raccoon dogs were collected. A total of 219 red foxes and 78 raccoon dog feces were tested for G. duodenalis cysts using the same methods as for cattle and domestic dogs. Information about age, hunting parish, and forestry districts where animals were hunted had been collected from hunters. Finally, the evaluation of the examined animal species across the three different ecosystems – rural, urban, and wildlife, contribution to the environmental contamination with G. duodenalis cysts was done. For this purpose, the cysts per gram (CPG) was compared between species, and then adjusted to the mean fecal/scat mass (grams per defecation), and number of defecations per day, to calculate the daily G. duodenalis cyst load to evaluate impact on the general environment. Additionally, the spatial distance between the zoonotic G.duodenalis assemblage A-positive animals and surface waterbodies was assessed to estimate a possible environmental spill-off. The doctoral thesis hypothesis: The prevalence, genetic diversity, and environmental cyst load of G. duodenalis differ among cattle (Bos taurus), domestic dogs (Canis familiaris), red foxes (Vulpes vulpes), and raccoon dogs (Nyctereutes procyonoides) in Latvia and are influenced by farming practices, housing conditions, and animal-specific factors. The aim of the doctoral thesis was to analyze the prevalence, cyst load, and genetic diversity of G. duodenalis in cattle (Bos taurus), domestic dogs (Canis familiaris), red foxes (Vulpes vulpes), and raccoon dogs (Nyctereutes procyonoides) in Latvia, to identify factors associated with the increased prevalence of G. duodenalis in studied animal species and to assess their potential contribution to environmental contamination. Tasks of the doctoral thesis: 1. to analyze the prevalence, cyst load, genetic diversity, animal-level and herd-level factors potentially associated with G. duodenalis in cattle; 2. to detect the prevalence, cyst load, genetic diversity, and animal-level and housing factors potentially associated with G. duodenalis in domestic dogs; 3. to establish the prevalence, cyst load, genetic diversity, and animal-level factors potentially associated with G. duodenalis in red foxes and raccoon dogs; 4. to assess and compare the cyst-shedding intensity of cattle and canids, to determine their contributions to environmental contamination with G. duodenalis zoonotic assemblage A. Scientific novelty of the doctoral thesis: 1. the first study in Latvia on the prevalence of G. duodenalis, cyst load, genetic diversity in cattle, domestic dogs, red foxes and raccoon dogs in Latvia, revealing G. duodenalis assemblages C, D and E and the zoonotic assemblage A; 2. identified factors associated with the prevalence of G. duodenalis in cattle, domestic dogs, red foxes and raccoon dogs in Latvia, providing veterinarians and experts in the field with insight into how to limit the spread of this parasite; 3. provides insight into the potential dissemination of zoonotic G. duodenalis assemblage A in the environment within the One Health approach. Authors’ personal contributions: 1. collection of feces from cattle; 2. communication with the herd owners and dog owners to collect fecal samples and conduct questionnaires; 3. feces preparation using immunofluorescence staining technique for G. duodenalis cyst detection and microscopy; 4. molecular analyses, including genomic G. duodenalis DNA isolation, PCR amplification, and Restriction Length Fragment Polymorphism Analysis (RFLP), were done personally in most cases; 5. descriptive statistics and risk and protective factor analysis using generalized linear mixed and generalized linear models to identify risk and protective factors associated with the prevalence of G. duodenalis in animal species were done personally in most cases. Giardia duodenalis prevalence in cattle in Latvia reached 8.4% (82/973) with the animals were shedding an average of 5756 CPG of feces. The highest prevalence of 16.4% (53/324) was observed in the 0–3-month-old cattle age group, followed by 6.8% (19/281) in the 4–24-month-old and 2.7% (10/368) in the > 24-month-old age groups. Significantly higher Giardia cyst load was shed by cattle in the 0–3-month-old age group (p = 0.0005). The highest proportion of diarrhea was observed in the 0-3-month-old cattle age group; but no statistical significance was observed between diarrhea and the presence of G. duodenalis (p > 0.05). Cattle herd prevalence was up to 84.4% (27/32) with the highest prevalence of 100% in herds with 251–500 (7/7) and more than 500 cattle (8/8), and 90.0% (9/10) in the herds with less than 150 cattle. The lowest prevalence of G. duodenalis was observed in herds with 151–250 cattle (42.9%, 3/7). In individual cattle, G. duodenalis assemblage A was detected in 11.3% (7/62), but assemblage E was detected in 88.7% (55/62) of the positive cattle. G. duodenalis assemblage A was identified in five cattle from the 4–24-month-old age group (71.4%, 5/7) and two cattle from the >24-month age group (28.6%, 2/7). G. duodenalis assemblage E was detected in 76.4% (42/55) of the calves from 0–3-month-old age group, followed by 14.5% (8/55) in the 4–24-month-old age group, and 9.1% (5/55) in the >24-month-old age group. For G. duodenalis-associated factors in cattle, in the final model, one risk (ability to leave the herd premises) and five protective factors (age, pasture season beginning in May or no pastures; manure kept in open pit or pile) appeared to be associated with the reduction of G. duodenalis infection in cattle in Latvia. The prevalence of G. duodenalis in domestic dogs was 10.7% (40/373), with the highest prevalence in the puppy age group (under one year old) (18.5%, 12/65). Significantly higher prevalence was observed in male dogs than female dogs (p = 0.01), but no differences were observed between sex and cyst load (p = 0.05). In domestic dogs, the zoonotic assemblage A was detected in 10.5% of the dogs (2/19), and dog-specific assemblages C and D were detected in 31.6% (6/19) and 42.1% (8/19) of the dogs, respectively. In the final GLM, G. duodenalis was associated with male dogs and co-infection with Cryptosporidium spp., but activity outside the city with a leash was a protective factor against G. duodenalis infection. In red foxes, G. duodenalis prevalence was 27.4% (60/219), with the highest prevalence observed in animals around five years old (50.0%, ½). No significant differences were observed among the age groups and cyst shedding in red foxes (p = 0.07). In raccoon dogs, the prevalence reached 30.8% (24/78), and the highest prevalence was observed in the 1–1.5-year-old age group (46.7%, 7/15). Differences between age and G. duodenalis cyst load were not significant (p = 0.7) in raccoon dogs. In red foxes, assemblages C and D were detected in one animal each, but one raccoon dog was positive for assemblage D. In the final GLM, the increased prevalence of G. duodenalis was associated with older animals (OR 2.3, p = 0.007) and co-infection with Cryptosporidium spp. (OR 111.1, p