Molecular Epidemiology of Cryptosporidium and Giardia as waterborne zoonoses

Abstract

Cryptosporidium spp. and Giardia spp. are two of the leading causal agents of parasitic diarrhoea in humans and calves. Both of the pathogens are host-adapted.There is a critical need to understand factors potentially associated with the risk and prevalence of infection due to Cryptosporidium spp. and Giardia spp. in humans and calves. Furthermore, molecular characterization of human and calves isolates may identify zoonotic genotypes and provide further information concerning the transmission routes between humans and calves. Thus, this study aimed to understand the epidemiology and molecular characterization of Cryptosporidium and Giardia in humans and animals in Chattogram, Bangladesh. In Chapter 2, a review of the literature regarding Cryptosporidium spp. and Giardia spp. in children and animals (calves) was conducted. The review involves a brief description of the two pathogens’ current taxonomy, epidemiology, transmission, and diagnostic methods. The thesis was organized with three objectives based on the epidemiological, molecular (nested PCR and real-time PCR) study of Cryptosporidium and Giardia from children and calves feces samples and metagenomics study was the comparison of the fecal microbiome of diarrhoeic and nondiarrhoeic calves samples and comparing the pathogens to see if there were any differences in the bacteriome community among them. The study was executed in the Chattogram Metropolitan area, which is situated in the south-eastern part of Bangladesh namely Chattogram, in districts denoted as plain and hilly regions, respectively, from January 2019 to January 2022, in different stages.

A cross-sectional survey was conducted in Chapter 3 to approximate the prevalence and associated risk factors of Cryptosporidium and Giardia species infections in children and calves in Chattogram. The study's primary goal was to ascertain the prevalence of these infections among these animals. A total of 437 (n = 437) fecal deposits from diarrheal people and calves were used in this investigation. 200 (n = 200) human fecal samples were taken from pediatric patients (ages 1 month to 12) who were referred to the Chattogram Medical College Hospital and had gastrointestinal distress, including diarrhea, dehydration, abdominal pain, nausea, and vomiting. Meanwhile, 237 (n = 237) fecal samples were also taken from calves ranging in age from one to six months, coming from various nearby farms in the Chattogram Metropolitan area.

All samples were stained with a modified Ziehl-Neelsen acid-fast for Cryptosporidia and Trichrome stain for Giardia. From the result of the fecal analyses to the modified Z-N stain, the prevalence of Cryptosporidium infection among hospitalized diarrheic children and infected calves was 13.5% and 23.63%, respectively. However, in SSUr RNA gene-based PCR, the prevalence of Cryptosporidium infection among hospitalized diarrheic children and infected calves was 9.5% and 19.41%, respectively. Additionally, trichrome staining revealed that the prevalence of Giardia in children and calves was 9.5% and 19.41%, respectively. While TPI gene-based PCR showed that 9% Giardia infection prevalence in children and 10.55% in calves. In humans, gender-based distributions for cryptosporidiosis and giardiasis indicated that males in cryptosporidiosis and giardiasis were at a higher risk for infection than females in cryptosporidiosis and giardiasis. Cryptosporidiosis was more prevalent in children aged 11-15 months, where as giardiasis was more frequent in children more than >35 months. The variation of seasons significantly influences the prevalence and incidence of cryptosporidiosis and giardiasis in the human population. Giardiasis was shown to be more likely to spread during the winter months, while cryptosporidiosis was more predominant in summer. In all scenarios, children residing in rural areas were more susceptible to infection in cryptosporidiosis and giardiasis than others residing in urban areas, including city and slum environments. Additionally, children who were deprived of proper sanitation were more susceptible to cryptosporidiosis and giardiasis. Water sources were also impacted on both kinds of infections, where children who consumed pond water rather than supplied and tube well water, were more prone to cryptosporidiosis and giardiasis. Children without a history of being breastfed are more susceptible to both types of infections. Nevertheless, there is no evidence to suggest that the historical record of pet ownership and exposure to anthelmintics has any influence on the occurrence of cryptosporidiosis and giardiasis in humans.The correlations between clinical characteristics and disease prevalence were also investigated and analyzed. The condition was not shown to be associated with a history of nausea, abdominal pain, anorexia, vomiting, or dehydration. Different variables with Cryptosporidium at the animal level were diagnosed by PCR and the frequency of cryptosporidiosis was found to be much higher in female calves compared to their male counterparts. The frequency of cryptosporidiosis was found to be relatively high in calves aged between 31 and 60 days. At the farm level, the frequency of cryptosporidiosis was shown to be independent of many factors, including the education level of owners, topography, types of calf housing, and floor type. Nevertheless, a substantial incidence of cryptosporidiosis was seen in calves residing in hilly regions and calves housed in enclosed barns with rubber pad flooring. Furthermore, it was shown that the incidence of cryptosporidiosis was comparatively elevated in calves that were exposed to ground water as opposed to pond or supply water sources. There was also an increase in the frequency of cryptosporidiosis among calves that did not share feeding utensils. Moreover, the frequency of cryptosporidiosis was higher in calves that were hand-fed compared to suckling-feeed.

In chapter 4, the second objective was to molecularly characterize of Cryptosporidium and Giardia in human and animal populations in the study area. The molecular prevalence of Cryptosporidium, among hospitalized diarrheic children and infected calves was found to be 9% (n=18) and 4.22% (n=10), respectively. On the other hand, the molecular prevalence of Giardia among hospitalized diarrheic children and infected calves was found to be 14% (n=28) and 10.55% (n=25), respectively. In terms of Cryptosporidium, the prevalence of the gp60 gene was determined to be 10% (n = 20) in hospitalized diarrheic youngsters and 11.39% (n = 27) in infected calves. The SSU gene was reported to be 6% (n=12) in hospitalized diarrheic children and 10.13% (n = 24) in infected calves. In the instance of Giardia, the prevalence of the TPI gene among hospitalized diarrhoeic children was reported to be 10.5% (n = 21), with no TPI gene detected in calves but bita-gardian found in calves only after PCR amplification. Quantitative real-time PCR (qPCR) was performed using an assay for detection of all Cryptosporidium and Giardia. A total number of 28 isolates were sequenced through Sanger sequencing approaches for both human and animal Cryptosporidium (gp60 = 14; SSU = 7) and Giardia (TPI = 7) isolates. The purified PCR products were applied for Sanger sequencing. Sequence quality was verified by comparison with corresponding electropherograms using the program Geneious v.8. Sequences were aligned using the program MUSCLE and alignments were adjusted manually using the program Mesquite v.2.75. Sequences were then compared with those available in the GenBank database using BLASTn. Firstly, the selected sequences were aligned using the Mafft algorithm, Phylogenetic analyses of the Cryptosporidium and Giardia were carried out based on gene-specific sequences such as (gp60, SSU, and TPI). Sequences were then validated in the GenBank database using BLASTn. However, after quality analysis, the sequences were stored in the NCBI nucleotide database through the following accession, OM665388.1 - OM665390.1, MT071440.1 - MT071443.1, OM877297.1 - OM877302.1, MT185587.1 - MT185589.1, OM877303.1 - OM877314.1.

Chapter-5: the third objective was the comparison of the fecal microbiome of diarrhoeic and non-diarrhoeic calves through metagenomics approach. This study was designed to unveil the gut bacteriome signatures and diversity by analyzing 10 samples including 5 diarrhoeic feces (DF) and 5 non-diarrhoeic feces (NDF) samples obtained from 10 individual calves through 16S rRNA (V4 region) gene-based amplicon sequencing. A total of 358 operational taxonomic units (OTUs) including 217 and 162 in DF and NDF samples, were identified. Findings revealed substantial taxonomic variability between sample categories (i.e., DF and NDF; p = 0.0127; Kruskal Wallis test) of the calves, indicated by their higher degree of shared microbiota. Of the identified genera, Gallibacterium (37.48%), Veillonella (14.53%), and Bacteroides (11.61%) were the major bacterial genera detected in the gut of calves. Importantly, we detected 44 genera including Sedimentibacter, Lonepinella, Sulfurospirillum, Haemophilus, Enterobacter, Citrobacter, etc. seem to be specific to calf diarrhea. Both the DF and NDF samples included 358 distinct bacterial species, of which 32.18% species were found to be shared between sample categories, and 25.14% and 11.73% species were found solely in DF and NDF, respectively. Moreover, Gallibacterium salpingitidis was found as the most prevalent species (43.37%) in DF samples followed by Gallibacterium anatis (17.56%), Bacteroides sp. (6.2%). In contrast, Veillonella magna had the highest prevalence (19.21%) in NDF samples followed by Bacteroides sp. (18.00%), Veillonella sp. (13.09%), and Ruminococcus sp. (7.23%). The findings suggested that diarrhoea affects the gut bacteriome in calves, with different microbial taxa associated with diarrhoea. Our data provided evidence for the existence of both unique and shared bacteriomes with pathogenic potentials in the gut of calves which might be taken into consideration for undertaking future microbiome studies in diarrhoeic calves.

As a whole, this PhD project provides new data on information of Cryptosporidium parvum infections in calves and Cryptosporidium hominis in children and also Giardia intestinalis in children. Results indicate that ruminants (calves) and children in Bangladesh shed potentially zoonotic pathogens in the environment and may contribute to the contamination of surface water. Our research in Bangladesh reveals a high prevalence of Cryptosporidium and Giardia, particularly affecting children and calves those are associated poor sanitation conditions. Molecular diagnostic techniques, such as PCR and Real-time PCR, have enhanced the identification and differentiation of these pathogens. Metagenomics studies have uncovered significant genetic diversity and multiple co-infections, offering deep insights into pathogen dynamics. Despite the advanced research tools, integrating these methods into public health practice remains challenging but we hope it paving the way for more effective control and prevention measures for the diarrheal diseases in our country.