Giardia duodenalis in Wildlife: Exploring Genotype Diversity in Italy and across Europe

Fragmented data are so far available on genotype diversity of G. duodenalis in wildlife in different countries in Europe, in particular, in Italy. In the present study, G. duodenalis sequences obtained from different Italian wild animals [12 porcupines (Hystrix cristata), 4 wild boars (Sus scrofa), 1 wolf (Canis lupus italicus), 6 Alpine chamois (Rupicapra rupicapra rupicapra)] were compared with those available from wild host species in Europe to add new data on the geographic distribution of Giardia assemblages/sub-assemblages and their transmission patterns among natural hosts. Thirty-eight sequences were obtained by MLG analysis (SSU-rRNA, bg, gdh, and tpi genes) and subsequently compared by phylogenetic and network analyses with those from wild species monitored in the last decades in Europe. The results revealed the presence of potentially zoonotic (A-AI, A-AII from wild boar; B from porcupine) and host-adapted (D from wolf; E, A-AIII from chamois) assemblages and sub-assemblages and represent the first report for Italian wild boar. The analysis did not find any evidence of spatial or host segregation for specific genetic variants, mostly shared between different hosts from different European countries. However, conflicting evidence was found in genotypic assignment, advocating for data improvement and new genomic approaches.

Literature Review: Coinfection in Young Ruminant Livestock—Cryptosporidium spp. and Its Companions

The protozoan Cryptosporidium parvum is one of the major causative pathogens of diarrhoea in young ruminants; therefore, it causes economic losses and impairs animal welfare. Besides C. parvum, there are many other non-infectious and infectious factors, such as rotavirus, Escherichia coli, and Giardia duodenalis, which may lead to diarrhoeic disease in young livestock. Often, more than one infectious agent is detected in affected animals. Little is known about the interactions bet-ween simultaneously occurring pathogens and their potential effects on the course of disease. In this review, a brief overview about pathogens associated with diarrhoea in young ruminants is presented. Furthermore, information about coinfections involving Cryptosporidium is provided.

Cryptosporidium and Giardia in Livestock in Tigray, Northern Ethiopia and Associated Risk Factors for Infection: A Cross-Sectional Study

The occurrence and species/genotypes of Cryptosporidium and Giardia duodenalis infecting young livestock in selected districts of Tigray, Ethiopia were investigated, along with risks associated with infection. A total of 757 faecal samples were collected from calves, lambs, and goat kids from four rural districts in Tigray, and also from calves in periurban Mekelle, Tigray's main city, and analysed for Cryptosporidium oocysts and Giardia cysts. Farmers answered questionnaires regarding potential risk factors at sample collection. Immunofluorescent antibody staining was used for parasite detection, and PCR at selected genes and sequencing of positive samples was used for molecular characterisation. The occurrence of Cryptosporidium infection was 10, 9, and 4% in calves, lambs, and goat kids, respectively; equivalent figures for Giardia infection were 39, 32, and 21%. Molecular characterisation of Cryptosporidium isolates revealed C. ubiquitum, subtype XIIa in all three host species; C. ryanae in calves and goat kids; C. andersoni and C. bovis were identified only in calves, and C. xiaoi was identified in lambs. For Giardia, Assemblage E predominated in all host species, but among calf isolates we also identified a few potentially zoonotic genotypes (assemblages A (AI) and Assemblage B). Periparturient care was shown to be a particularly relevant risk factor for infection, and infections were less likely to occur under extensive management systems. Our major findings were widespread occurrence of both parasites in livestock, and the apparent lack of the most common zoonotic species. Our results are discussed in relation to other relevant studies. As our study was conducted in Tigray, further investigation in different settings in Ethiopia could provide relevant information on transmission and zoonotic potential. In addition, given the dependency on healthy animals for the livelihoods of the population of Tigray, investigation of the effect of these common parasites on livestock productivity is important.

Intestinal Protists in Captive Non-human Primates and Their Handlers in Six European Zoological Gardens. Molecular Evidence of Zoonotic Transmission

We assessed the occurrence, genetic diversity, and zoonotic potential of four protozoan (Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Giardia duodenalis), one stramenopile (Blastocystis sp.), one microsporidia (Enterocytozoon bieneusi), and two ciliate (Balantioides coli, Troglodytella abrassarti) intestinal parasite or commensal protist species in captive non-human primates (NHP) and their zookeepers from six European zoological gardens in France (n = 1), Germany (n = 1), and Spain (n = 4). Faecal samples from NHP (n = 454) belonging to 63 species within 35 genera and humans (n = 70) were collected at two sampling periods in each participating institution between October 2018-August 2021. Detection and species identification was accomplished by PCR and Sanger sequencing of the ssu rRNA and/or ITS genes. Sub-genotyping analyses using specific markers were conducted on isolates positive for G. duodenalis (gdh, bg, tpi) and Cryptosporidium spp. (gp60). Overall, 41.0% (186/454) and 30.0% (21/70) of the faecal samples of NHP and human origin tested positive for at least one intestinal protist species, respectively. In NHP, Blastocystis sp. was the most prevalent protist species found (20.3%), followed by G. duodenalis (18.1%), E. dispar (7.9%), B. coli and T. abrassarti (1.5% each), and Cryptosporidium spp. and E. bieneusi (0.9% each). Occurrence rates varied largely among NHP host species, sampling periods, and zoological institutions. The predominant protist species found in humans was Blastocystis sp. (25.7%), followed by Cryptosporidium spp. (2.9%), E. dispar (1.4%), and G. duodenalis (1.4%). Sequencing of PCR-positive amplicons in human and/or NHP confirmed the presence of Cryptosporidium in six isolates (C. hominis: 66.7%, C. parvum: 33.3%), G. duodenalis in 18 isolates (assemblage A: 16.7%, assemblage B: 83.3%), Blastocystis in 110 isolates (ST1:38.2%, ST2:11.8%, ST3: 18.2%, ST4: 9.1%, ST5: 17.3%, ST8: 2.7%, ST13: 0.9%), and E. bieneusi in four isolates (CM18: 75.0%, Type IV: 25.0%). Zoonotic transmission events involving Blastocystis ST1–ST4 were identified in four zoological institutions. Zoonotic transmission of C. hominis was highly suspected, but not fully demonstrated, in one of them. Monitoring of intestinal protist species might be useful for assessing health status of captive NHP and their zookeepers, and to identify transmission pathways of faecal-orally transmitted pathogens.

Prevalence, Risk Factors and Genotypes of Giardia duodenalis in Sheltered Dogs in Tuscany (Central Italy)

In sheltered dogs, the prevalence of Giardia duodenalis is frequently high and may include potential zoonotic genotypes. The prevalence, genotypes and potential risk factors of G. duodenalis were assessed in 168 dogs from four kennels (Pistoia, Prato, Florence, Valdarno) in Tuscany, central Italy and compared with data from previous Italian studies. The prevalence of other intestinal parasites was also investigated. Individual dog faecal samples collected from each kennel were examined by parasitological techniques and a rapid immunoassay for the detection of G. duodenalis and Cryptosporidium faecal antigens. On Giardia-positive samples, molecular analysis was performed for genotype identification. Overall, 69 dogs scored positive for G. duodenalis (41%), but significant differences (p ≤ 0.05) were found among the four kennels and sampling seasons. The potentially zoonotic assemblages A and B and the canine-specific assemblage C (Pistoia: A-AII, B, C; Prato: A-AII, B; Florence: A-AII; Valdarno: A and C) were identified. Toxocara canis (8.9%), Trichuris vulpis (3.6%), hookworms (1.19%) and Cryptosporidium sp. (0.6%) were also identified. The high prevalence of G. duodenalis and the identification of potentially zoonotic genotypes in all examined kennels underline the need to improve routine parasite monitoring and control measures and to provide insights into the zoonotic potential of G. duodenalis.

Prevalence and Molecular Characterization of Giardia duodenalis Assemblage D of Dogs in Egypt, and Its Zoonotic Implication

Background, Methods: To elucidate the prevalence and molecular characterization of Giardia infection in dogs, a cross-sectional study was performed on stray and housed dogs from different localities of Egypt. Results: A total of 986 fecal specimens were collected from dogs. The overall infection rate was 8.5%. The diarrheic dogs revealed higher prevalence of 14.43% than asymptomatic animals. The infection was more prevalent in younger dogs ( < 6 months) (9.5%) compared to older animals. Higher prevalence of infection was observed during the cold winter months (11.24%). The community owned dogs (stray dogs in the street) showed (11.75%) higher prevalence rate than pet dogs in the household environment (5.59%). Moreover, it was found that dogs fed on undercooked meat, and offal's were showing higher prevalence of giardiasis than dogs fed on canned meat. On the other hand, dogs subjected to regular grooming and good hygienic practices had lesser prevalence rate of the infection compared to unclean neglected dogs. Conclusions: The sequencing and phylogenetic analysis of the amplicons of 18SrRNA gene of G. duodenalis revealed that; they were closer to assemblage D necessitating urgent attention due to their zoonotic importance.

The Prevalence and Risk Factors of Giardia duodenalis Infection in Cats in Mexico

Aims: Giardia duodenalis is a globally distributed zoonotic protozoan. It has a variable prevalence. This study determines the prevalence of Giardia spp. in cat faecal samples from states of the Mexican Republic. Place and Duration of Study: Was carried out in 23 of the 32 states of the Mexican Republic, from June to December 2019. Methodology: Stool samples from 1591 client-owned cats were analysed for the detection of G. duodenalis (cysts or trophozoites). Faecal samples were analysed by direct smear techniques with and without staining (Lugol) and centrifugal floatation (faust), and were examined under a light microscope. Results: Of the cats sampled, 56.94% were positive for G. duodenalis. Its prevalence was associated and is a risk factor in cats that live with other animals (Chi2= 21.84, p= 0.0001; OR= 1.61, p= 0.0001), with hunting habits (Chi2= 5.53 p= 0.01, OR= 1.27 p= 0.01), with access to the outside (Chi2= 53.06, p= 0.0001; OR= 2.13, p=0.0001) and with the aqueous faeces (Chi2= 12.30, p=0.03; Chi2= 1.71, p= 0.03). Factors for not presenting Giardia spp. in faeces were, not brushing the cat (OR=0.74, p= 0.006), provenance (OR= 0.42, p=0.02), and median height (OR= 0.78, p= 0.01). Age, gender, hair type, coexistence with other cats and other stool findings were not associated as risk factors for infection. Conclusion: This study demonstrated a high overall prevalence of G. duodenalis in cats in Mexico, in addition to an association of its prevalence with risk factors such as cats living with other animals, hunting habits and access to outdoors.

Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.