Infection rate and genetic diversity of Giardia duodenalis in pet and stray dogs in Henan Province,China

Giardia duodenalis is an important protozoan parasite that is known to be zoonotic. To assess the potential zoonotic transmission of giardiasis from dogs and to identify genetic diversity of G. duodenalis in dog populations, we examined the infection rate and genotypes of G. duodenalis in both pet dogs (from pet dog farms, pet shops, pet hospitals, pet markets) and stray dogs of different ages in Henan Province, China. A total of 940 fresh fecal specimens were collected from 2007 to 2013 in Henan Province. The overall infection rate of G. duodenalis was 14.3% (134/940) as determined by microscopy, with the highest infection rate (17.3%) observed in dogs from shelters. Young dogs were more likely to be infected with G. duodenalis than adult dogs, and G. duodenalis cysts were found more frequently in diarrheic dogs. All G. duodenalis-positive isolates were characterized at the triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) loci, and 37, 51, and 48 sequences were obtained, respectively. The dog-specific assemblages C and D were identified using multi-locus sequence analysis. Six novel sequences of the tpi locus, one novel sequence of the gdh locus and two novel sequences of the bg locus were detected among the G. duodenalis assemblage C isolates, while two novel sequences of the gdh locus were found among the G. duodenalis assemblage D isolates. Our data indicate that G. duodenalis is a common parasite and cause of diarrheal disease in dogs in Henan Province. However, there was no evidence for zoonotic G. duodenalis assemblages in the study population.     

Effects of anthropogenic habitat disturbance and Giardia duodenalis infection on a sentinel species' gut bacteria

Habitat disturbance, a common consequence of anthropogenic land use practices, creates human–animal interfaces where humans, wildlife, and domestic species can interact. These altered habitats can influence host–microbe dynamics, leading to potential downstream effects on host physiology and health. Here, we explored the effect of ecological overlap with humans and domestic species and infection with the protozoan parasite Giardia duodenalis on the bacteria of black and gold howler monkeys (Alouatta caraya), a key sentinel species, in northeastern Argentina. Fecal samples were screened for Giardia duodenalis infection using a nested PCR reaction, and the gut bacterial community was characterized using 16S rRNA gene amplicon sequencing. Habitat type was correlated with variation in A. caraya gut bacterial community composition but did not affect gut bacterial diversity. Giardia presence did not have a universal effect on A. caraya gut bacteria across habitats, perhaps due to the high infection prevalence across all habitats. However, some bacterial taxa were found to vary with Giardia infection. While A. caraya's behavioral plasticity and dietary flexibility allow them to exploit a range of habitat conditions, habitats are generally becoming more anthropogenically disturbed and, thus, less hospitable. Alterations in gut bacterial community dynamics are one possible indicator of negative health outcomes for A. caraya in these environments, since changes in host–microbe relationships due to stressors from habitat disturbance may lead to negative repercussions for host health. These dynamics are likely relevant for understanding organism responses to environmental change in other mammals.     

Antigenic analysis of Giardia duodenalis strains isolated in Alberta

Giardia duodenalis is a common intestinal parasite in most parts of the world. In Canada it is associated with both endemic and epidemic infections that are often transmitted by the waterborne route. Although G. duodenalis strains have been isolated from several animals, the role of other mammals in human infection is unclear. We have isolated and cultured G. duodenalis trophozoites from domestic and wild animals in Alberta and compared them with a human isolate by protein gel electrophoresis and immunoblot analysis. All strains examined share a similar polypeptide profile and important protein antigens. Prominent antigens of 62, 52, 38, and 31 kilodaltons are conserved. The 52- and 31-kilodalton proteins are the major surface-exposed trophozoite components. The high degree of antigenic sharing among strains from different hosts suggests that there may be a wide range of potential reservoirs for G. duodenalis infections.     

Genetic Exchange Within and Between Assemblages of Giardia duodenalis

ABSTRACT. Meiotic sex evolved early in the history of eukaryotes. Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis ), a parasitic protist belonging to an early diverging lineage of eukaryotes, shows no cytological or physiological evidence of meiotic or sexual processes. Recent molecular analyses challenge the idea that G. duodenalis is a strictly clonal organism by providing evidence of recombination between homologous chromosomes within one subgroup (Assemblage A) of this species as well as genetic transfer from one subgroup to another (Assemblage A–B). Because recombination is not well documented and because it is not known whether the observed inter-assemblage transfer represents true reciprocal genetic exchange or a non-sexual process, we analyzed genic sequences from all major subgroups (Assemblages A–G) of this species. For all assemblages, we detected molecular signatures consistent with meiotic sex or genetic exchange, including low levels of heterozygosity, as indicated by allelic sequence divergence within isolates, and intra- and inter-assemblage recombination. The identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex.     

Multilocus genotyping of Giardia duodenalis from pigs in Korea

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is an important zoonotic parasite infecting livestock (including pigs) through ingesting cysts in contaminated food or water. This parasite has been classified into eight different genetic assemblages, A to H. Here, we examined the individual-level prevalence of G. duodenalis in domestic pig farms and confirmed host specificity by genotype comparisons. Samples were collected from southern and central Korea, between May 2017 and January 2019. DNA directly extracted from 745 pig fecal specimens were tested by PCR for G. duodenalis small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), and β-giardin gene sequences. Based on ssu rRNA PCR, 110 (14.8%) were positive for G. duodenalis. Infection risk was the highest in the fattener group (31/139, 22.3%) and during the autumn season (52/245, 21.2%: p < .001). No statistically significant differences in risk for infection were observed between fecal types (normal versus diarrheal). Fifty ssu rRNA samples, three gdh samples, and five β-giardin samples were successfully sequenced and genotyped. Ssu rRNA assemblage sequence analysis identified E (40.0%, 20/50), D (34.0%, 17/50), C (24.0%, 12/50), and A (2.0%, 1/50). The gdh locus identified three samples as assemblage E, and the β-giardin locus identified four samples as assemblage E and one as assemblage C. Assemblage A sequences obtained (ssu rRNA; MK430919) had 100% identity with Giardia sequences isolated from a Korean individual (AJ293301), indicating the potential of zoonotic transmission. Continuous management and monitoring for prevention of transmission and protection of animal and human health are essential.     

Mixed Giardia duodenalis assemblage A and E infections in calves

A molecular epidemiological study was conducted on 100 dairy (499 calves) and 50 beef (333 calves) farms in Belgium to estimate the prevalence of different Giardia duodenalis assemblages in calves younger than 10 weeks of age. Positive samples from the epidemiological study and from a previous clinical study were selected and genotyped based on the amplification of the beta-giardin gene. To investigate the occurence of mixed assemblage A and E infections in calves, a novel assemblage-specific PCR was developed based on the triose-phosphate isomerase gene. The prevalence was 22% (95% Probability Interval (PI): 12-34%) in dairy calves and 45% (95% PI: 30-64%) in beef calves. In total, 120 Giardia-positive samples from dairy and beef calves collected in the epidemiological study and from clinically affected calves were identified based on the amplification of the beta-giardin gene. Overall G. duodenalis assemblage E was more prevalent (in 64% of the samples), although the majority (59%) of the dairy calves were infected with G. duodenalis assemblage A. Furthermore, mixed G. duodenalis assemblage A and E infections were identified in 31% of the calf samples (n=101) using the assemblage-specific PCR. We believe this is the first report of mixed infections in calves, and the results of the present study indicate that calves, although mainly infected with the host-specific G. duodenalis assemblage E, are frequently infected with the zoonotic assemblage A, either as a mixed or mono-infection, suggesting that calves might be underestimated as a potential zoonotic reservoir for human infections.     

Identification of nucleoli in the early branching protist Giardia duodenalis

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.     

Projections of the Current and Future Disease Burden of Hepatitis C Virus Infection in Malaysia

Background

The prevalence of hepatitis C virus (HCV) infection in Malaysia has been estimated at 2.5% of the adult population. Our objective, satisfying one of the directives of the WHO Framework for Global Action on Viral Hepatitis, was to forecast the HCV disease burden in Malaysia using modelling methods.

Methods

An age-structured multi-state Markov model was developed to simulate the natural history of HCV infection. We tested three historical incidence scenarios that would give rise to the estimated prevalence in 2009, and calculated the incidence of cirrhosis, end-stage liver disease, and death, and disability-adjusted life-years (DALYs) under each scenario, to the year 2039. In the baseline scenario, current antiviral treatment levels were extended from 2014 to the end of the simulation period. To estimate the disease burden averted under current sustained virological response rates and treatment levels, the baseline scenario was compared to a counterfactual scenario in which no past or future treatment is assumed.

Results

In the baseline scenario, the projected disease burden for the year 2039 is 94,900 DALYs/year (95% credible interval (CrI): 77,100 to 124,500), with 2,002 (95% CrI: 1340 to 3040) and 540 (95% CrI: 251 to 1,030) individuals predicted to develop decompensated cirrhosis and hepatocellular carcinoma, respectively, in that year. Although current treatment practice is estimated to avert a cumulative total of 2,200 deaths from DC or HCC, a cumulative total of 63,900 HCV-related deaths is projected by 2039.

Conclusions

The HCV-related disease burden is already high and is forecast to rise steeply over the coming decades under current levels of antiviral treatment. Increased governmental resources to improve HCV screening and treatment rates and to reduce transmission are essential to address the high projected HCV disease burden in Malaysia.     

Occurrence of Giardia duodenalis assemblages in alpacas in the Andean region

In this study, 352 fecal samples were analyzed for G. duodenalis from alpaca mothers and crias from three different areas of highland in Peru. The triosephosphate isomerase (TPI) gene of Giardia was amplified using a nested PCR protocol. Forty-six G. duodenalis-PCR positive samples were sequenced. G. duodenalis assemblage A was the most frequent followed by assemblage E. The former was seen in 37 animals whereas the latter was seen in nine. Most of the assemblage A infections were caused by the A1 subtype of sub-assemblage AI, except for three, which were caused by the A2 subtype of sub-assemblage AI. Assemblage A was found in all three geographic regions, while assemblage E was detected in crias from two regions. Among the four alpaca mothers positive for Giardia, three had assemblage AI and one had assemblage AII. Results of this study indicate that possible zoonotic transmission human to alpacas.     

Influence of refrigeration and formalin on the floatability of Giardia duodenalis cysts.

Giardia duodenalis cysts obtained from fresh fecal samples, fecal samples kept under refrigeration and fecal samples treated with formalin were studied as to their floatability on sucrose solutions with the following specific gravities: 1,040 kg/m3; 1,050 kg/m3; 1, 060 kg/m3; 1,070 kg/m3; 1,080 kg/m3; 1,090 kg/m3; 1,100 kgm3; 1,150 kg/m3; 1,200 kg/m3; and 1,250 kg/m3, contained within counting-chambers 0.17 mm high. Cysts that floated on and those settled down as sediments were counted, and had their percentages estimated. Sucrose solutions of 1,200 kg/m3 specific gravity (the average specific gravity of diluting liquids employed in floatation techniques) caused to float 77.7%, 78.4% and 6.6% of the G. duodenalis cysts obtained, respectively, from fresh fecal samples, fecal samples kept under refrigeration, and fecal samples treated with formalin. Cysts obtained both from fresh fecal samples and fecal samples kept under refrigeration presented similar results concerning floatability. It was observed, however, that the treatment of feces with formalin diminished the cysts floatability under the various specific gravities studied. This results should influence, the recommendations for transport and storage of fecal samples used for parasitological coproscopy.     

Nucleotide Variation in the Cytidine Triphosphate Synthetase Gene of Giardia duodenalis

ABSTRACT. The cytidine triphosphate synthetase genes from three diverse strains of Giardia duodenalis have been sequenced and found to vary significantly from one another. The isolates were chosen as representatives of three demes as determined by several criteria including divergence in the rDNA repeat unit. Inserts in the genes and protein are conserved in length but are the most divergent regions among the three sequences examined. Variation in the rest of the gene occurs primarily in the third base position resulting in many silent mutations. One of the isolates (1709) was found to contain two genes with high sequence homology.     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.     

Age patterns of Cryptosporidium species and Giardia duodenalis in dairy calves in Egypt

Little is known of the occurrence and age patterns of species/genotypes and subtypes of Cryptosporidium spp. and Giardia duodenalis in calves in Egypt. In this study, 248 fecal specimens were collected from dairy calves aged 1?day to 6?months on eight farms in three provinces during March 2015 to April 2016. Cryptosporidium spp. were detected and genotyped by using PCR-RFLP analysis of the small subunit rRNA (SSU rRNA) gene, while G. duodenalis was detected and genotyped by using PCR and sequence analyses of the triose phosphate isomerase (tpi), glutamate dehydrogenase (gdh) and β-giardin (bg) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 9.7 and 13.3%, respectively. The highest Cryptosporidium infection rate (26.7%) was in calves of age?≤?1?month while the highest G. duodenalis infection rate (44.4%) was in calves of 2?months. Three Cryptosporidium spp. were identified, including C. parvum (n?=?16), C. bovis (n?=?5) and C. ryanae (n?=?3), with the former being almost exclusively found in calves of ≤3?months of age and the latter two being only found in calves of over 3?months. Subtyping of C. parvum by PCR-sequence analysis of the 60?kDa glycoprotein gene identified subtypes IIaA15G1R1 (n?=?15) and IIaA15G2R1 (n?=?1). The G. duodenalis identified included both assemblages E (n?=?32) and A (n?=?1), with the latter belonging to the anthroponotic subtype A2. These data provide new insights into the genetic diversity and age patterns of Cryptosporidium spp. and G. duodenalis in calves in Egypt.     

Immune and pathophysiological responses to different strains of Giardia duodenalis in neonatal mice

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti-Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia. This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.     

Immunoblot patterns of Giardia duodenalis isolates from different hosts and geographical locations

Eighteen isolates of Giardia duodenalis from animal and human sources were studied for protein differences by polyacrylamide gel electrophoresis and for antigenic differences by immunoblot analysis. The polyacrylamide gels showed that whilst the isolates were for the most part homogeneous in their protein banding patterns, some isolates did show some differences. The immunoblot analysis yielded many bands, including prominent bands of 32 and 66 kilodaltons. Five of the six isolates that showed differences in protein banding pattern also showed differences in antigenic reactivity. Our findings suggest that differences can be seen with the use of immunoblotting and that this technique is a tool that may be useful for isolate differentiation when used in conjunction with other techniques.     

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in experimental rats in China

Cryptosporidium and Giardia are ubiquitous protozoan parasites that infect a broad range of hosts. The presence of Cryptosporidium spp. and G. duodenalis was detected in 355 fecal samples of laboratory experimental rats from four experimental rat rearing facilities in China by PCR amplification of the small subunit (SSU) rRNA gene. The G. duodenalis positive samples were further characterized in the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 0.6% (2/355) and 9.3% (33/355), respectively, with no co-infection. Among the four facilities, only the rats in Zhengzhou1 were found positive for the two pathogens. Undetermined Cryptosporidium genotype was observed in one sample and C. ubiquitum in another sample. Assemblage G was identified in all the 33 G. duodenalis positive isolates at SSU rRNA gene, out of which 19, 20, and 21 isolates were also subtyped as assemblage G at tpi, gdh and bg gens, respectively. To our knowledge, this is the first report of Cryptosporidium and G. duodenalis infections in laboratory experimental rats in China. The infections of these pathogens in laboratory animals should be monitored routinely since they may interfere the biological experiments in these animals.