Prevalence and molecular characterization of Cryptosporidium species in poultry in Bangladesh.

Cryptosporidium is an opportunistic parasite that has been reported in >30 avian hosts worldwide, however, there is no information regarding Cryptosporidium spp. in poultry in Bangladesh. Accordingly, we investigated the prevalence of Cryptosporidium spp. in poultry at open live bird markets in Bangladesh. A total of 197 samples were randomly collected from poultry at open live bird markets in Bangladesh and screened for the detection of Cryptosporidium. Initial microscopic examination revealed Cryptosporidium spp. was observed in 19.8% (39/197) of the poultry specimens. Subsequent nested PCR targeting the 18S rRNA gene revealed that 15.7% (31/197) of the samples were Cryptosporidium positive. Of these 31 samples, 17 were Cryptosporidium baileyi (8.7%), 12 were Cryptosporidium meleagridis (6.0%), and 2 were Cryptosporidium parvum (1.0%). Nucleotide sequence analysis of the GP60 gene of the C. meleagridis revealed that two subtypes (IIIbA21G1R1 and IIIbA23G1R1), which were found in broiler, native and sonali chickens and a pigeon, matched those previously reported in humans and poultry. We identified two novel subtypes (IIIbA21G2R1 and IIIbA20G2R1) in sonali chickens, a broiler chicken and a layer chicken. We also amplified the GP60 gene of C. parvum and found two subtypes (IIaA11G2R1 and IIaA13G2R1) in a sonali and a broiler chicken that were previously reported in calf. These findings suggest that poultry can be a source of cryptosporidial infections for humans and animals in Bangladesh. This is the first molecular investigation of Cryptosporidium genotypes and subtypes in poultry at open live bird markets in Bangladesh.

Introduction

Cryptosporidiosis is a protozoan disease widely found in wild, domestic, and captive birds from several parts of the world [36]. Cryptosporidium has been recorded in >30 avian species worldwide, including chickens, turkeys, ducks, geese, quails, pheasants, and peacocks [31]. So far, infections in birds are mainly caused by four species: C. baileyi, C. galli, C. meleagridis, and C. avium [29]. Cryptosporidium is transmitted through the fecal-oral route by environmentally resistant oocysts that are shed in the feces, contaminating soil and water, and thus providing multiple pathways into the food chain [32]. C. meleagridis is the only Cryptosporidium species known to infect both birds and mammals [5]. Therefore, humans can acquire cryptosporidiosis either by consumption of water and food contaminated with oocysts or direct contact with infected people or animals [49]. In 2010, C. baileyi was reported to be the dominant Cryptosporidium species found in all age groups of chickens [44], and in 2017, C. parvum was the most frequently observed species identified in poultry in Germany [14]. Cross-species transmission of C. meleagridis between birds and humans has been reported in Peru [43,45]. Cryptosporidium meleagridis is considered the third most common species of this genus emerging in humans [47]. Cryptosporidium-infected domestic pigeons have been reported in some regions of the world, such as in Thailand [19], China [23], and Brazil [30]. The markets where poultry is purchased are crowded with people, and it is possible for Cryptosporidium to spread to the surrounding environment due to close contact with other species of birds or mammals in the markets [4].

The poultry industry of Bangladesh is a promising sector for economic growth. In Bangladesh, documented studies of cryptosporidiosis in animals and poultry are limited. Moreover, no studies examining protozoan infections have been done in the open live bird markets of Bangladesh. A few studies of Cryptosporidium related to human disease have been done in Bangladesh, including reports of C. parvum and C. meleagridis in children in Bangladesh [15,20]. C. meleagridis was the predominant species in rural areas, whereas in urban areas C. meleagridis was the second most frequently identified species in Bangladesh after C. parvum [37].

In Bangladesh, open live bird markets offer wholesale poultry. Peoples gather to buy the poultry for consumption and sellers slaughter and process the poultry under unhygienic conditions. The poultry excrete droppings, which are not regularly cleaned up, creating a high risk for transmission of Cryptosporidium infection from poultry to humans. Therefore, the present study sought to determine the epidemiology of Cryptosporidium spp. in poultry at open live bird markets in Bangladesh and to molecularly characterize the Cryptosporidium spp. identified.

Materials and methods

Ethics statement

All samples were collected from live bird markets. Since samples were collected after slaughter of the poultry, no permission regarding laws on animal protection was required. We received permission from the poultry sellers to collect the samples.

Sample collection

A total of 197 intestinal colon samples from different poultry species including layer chickens (n = 12), broiler chickens (n = 80), sonali chickens (n = 93), native chickens (n = 8) and pigeons (n = 4) were obtained from 19 different live bird markets (LBM) in Dhaka, Bangladesh. The age of the poultry ranged from one month to two years. Poultry were kept together in cages for sale to customers. Fresh intestinal colon samples were collected after the slaughter of every bird from a cage, taking care to collect only the portion that did not have direct contact with the cage to avoid contamination. All relevant data such as breed and age were recorded for further analysis. Each sample was placed into an individual sterile polystyrene tube, labelled, and transferred in an isothermal box to the laboratory where it was kept at 4 °C until DNA extraction.

Microscopic examinations

The samples were examined for Cryptosporidium oocysts by using the sucrose flotation technique. Briefly, 1 g of colon contents from the sample was suspended in 9 ml of saturated sucrose solution (Specific gravity 1.2) in a tube and centrifuged at 1300g for 5 min. Then, sucrose solution was added up to the 15-ml tube, which was then left at room temperature for 30 min. Then, the tube was filled to create a meniscus by adding drops of solution and putting a coverslip over the tube for 5–10 min. After that, the coverslip was put on a glass slide and examined for oocysts under a light microscope [9]. Thereafter, oocysts of Cryptosporidium-positive samples were transferred from the surface of the suspension to a microscopic slide, air dried, and then fixed for 3–5 min in absolute methanol before they were stained with Ziehl–Neelsen carbol fuchsin for 20 min. After washing with tap water, smears were decolorized with 5% acid alcohol for 20–30 s, washed again with tap water and counterstained with 0.4% methylene blue for 2 min, then rinsed again with tap water and air dried. After drying, the stained smears were examined for oocysts under light microscope using an oil immersion objective lens (×100) [50]. The remaining samples were kept at 4 °C until DNA extraction [7].

DNA extraction

DNA was extracted directly from samples by using the QIAamp Stool Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions with minor modifications. Modifications included the addition of 0.2 g of E-Z zirconia beads to 0.2 g of feces and 1.4 ml of lysis buffer [27]. Then, the mixture was heated at 95 °C for 10 min followed by vigorous shaking using the Biomedicals Fast Prep® instrument (1620 r/min for 150 s) to facilitate oocyst rupture. Nucleic acid was eluted in 100 μl of AE buffer to increase the quantity of DNA recovered. After the DNA was extracted, it was stored at −20 °C.

Nested PCR analysis of the 18S rRNA and GP60 genes

For the primary PCR, a PCR product of 1325 bp was first amplified using the primers (Table S1). The amplification was performed in a 25-μl volume with 2 μl of each DNA sample in 12.5 μl of 2 × PCR buffer, 5 μl of deoxynucleotide triphosphates (2 mM each), 0.25 μl of each primer (50 μM), 4.5 μl of double distilled water, and 0.5 μl of KOD- Fx Neo amplification enzyme (1 units/μl) (ToYoBo Co., Ltd., Osaka, Japan). Then, for the nested-PCR (nPCR), 2 μl of the primary PCR product was used with appropriate primers to amplify a ~ 830-bp fragment of the Cryptosporidium 18S rRNA gene as described previously [48]. The PCR reaction consisted of an initial heating at 94 °C for 2 min, and 35 cycles of 98 °C for 10 s, 55 °C for 60 s, and 68 °C for 30 s (primary PCR) or 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min 30 s (nested PCR). The 60-kDa glycoprotein (GP60; ~ 830 bp; [2,11,24]) was also amplified in positive samples on the basis of the results for the 18S rRNA. For subtyping of C. meleagridis, nPCR with a specific set of primers was performed to amplify a 1100-bp and a 900-bp fragment of the GP60 gene [38]. For subtyping of C. parvum, nPCR with a specific set of primers was performed to amplify an 850-bp and an 800-bp fragment of the GP60 gene [8]. Primary PCR was carried out in a total volume of 25 μl with 2 μl of each DNA sample in 12.5 μl of 2 × PCR buffer, 5 μl of deoxynucleotide triphosphates (2 mM each), 0.25 μl of each primer (50 μM), 4.5 μl of nuclease-free water, and 0.5 μl of KOD- Fx Neo amplification enzyme (1 units/μl). For the nested PCR, 2 μl of the primary PCR product was used. The PCR and cycling conditions were unique to the primary and nPCR and consisted of an initial denaturation at 94 °C for 2 min, and 35 cycles of 98 °C for 10 s, 50 °C for 30 s, and 68 °C for 60 s. As positive and negative controls, C. parvum genomic DNA and ultrapure water, respectively, were used instead of sample DNA. Known positive standards were used during each PCR run. Optimization of PCR was achieved and different temperature and PCR run conditions were followed as described previously by other investigators who have used similar primer sets [8] to identify and characterize Cryptosporidium. The amplified fragments were electrophoresed in 1.5% agarose, stained with GelRed® (Biotium), and visualized on an UV transilluminator by electrophoresis in the QIAxcel Advanced system (Qiagen, Valencia, California).

Sequencing

PCR products were purified from 1.5% agarose gel by using NucleoSpin® gel and a PCR clean-up kit (MACHEREY-NAGEL, Germany). Amplicons of 18S rRNA and GP60 genes were directly sequenced in both directions with the primers used for the secondary PCR by the ABI 3100 Genetic Analyzer and the BigDye Terminator v3.1 Cycle Sequencing Kit. DNA sequences were assembled with Codoncode Aligner version 7.1.1 software (CodonCode Corporation). The consensus sequences were assembled with homologous sequences published in GenBank using Clustal W [42] and BioEdit Sequence Alignment Editor [12]. The acquired sequences were submitted to a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to initially define the species/genotypes and to confirm the high similarity and homology with other known sequences of Cryptosporidium spp. in GenBank. All sequences were multiple-aligned and analyzed by Bioedit and MEGA 7.0 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html and http://www.megasoftware.net/). Phylogenetic trees were generated using Maximum Likelihood (ML) analysis based on the T92 + I model [40] for 18S rRNA gene in MEGA7 [21] using Plasmodium falciparum as an out-group. Phylogenetic trees were also generated using ML analysis based on the Tamura-Nei model [41] for the GP60 genes of the Cryptosporidium subtypes in MEGA7 [21]. To assess the reliability of this tree, bootstrap analysis was done with 1000 replicates.

Nucleotide sequence accession number

The partial 18S rRNA nucleotide sequences and GP60 sequences obtained in this study have been deposited in the GenBank database under accession numbers (MN133966 - MN133996 and MN192414 - MN192426).

Statistical analyses

Data analysis was performed using Microsoft Office Excel 2010, and results were considered to be statistically significant when P-values were ≤ 0.05. Prevalence rates with 95% confidence intervals were calculated by using Wilson (score) intervals [34] obtained in OpenEpi software (http://www. openepi.com/Menu/OE_Menu.htm).

Results

Positive rates of Cryptosporidium spp. in poultry

Microscopic examinations revealed the presence of Cryptosporidium oocysts in 19.8% (39 of the 197) poultry samples (Fig. 1). The modified Ziehl-Neelsen method indicated that Cryptosporidium was present in 50% (2/4) of the pigeon samples, 20.4% (19/93) of the sonali chicken samples, 18.8% (15/80) of the broiler chicken samples, 16.7% (2/12) of the layer chicken samples and 12.5% (1/8) of the native chicken samples. Not all microscope-positive samples were PCR-positive. The positive rate for Cryptosporidium was 15.7% (31/197) by nested-PCR. The highest rate (50%) of Cryptosporidium was found in pigeons both microscopically and by PCR, whereas the lowest rate was found in the native chicken samples (12.5%) by the microscopic method and in broiler chickens (11.3%) by nPCR (Table 1).

Fig. 1

Microscopic observation of Cryptosporidium oocysts. Oocysts were observed by using the Ziehl-Neelsen staining method under a microscope.

Table 1

Positive rates of Cryptosporidium species in poultry.

Poultry spp.	No. sample	No. microscopy positive (%)	No. nested PCR positive (%)
Chicken (sonalis)	93	19 (20.4)	17 (18.3)
Chicken (Broilers)	80	15 (18.8)	9 (11.3)
Chicken (layers)	12	2 (16.7)	2 (16.7)
Chicken (natives)	8	1 (12.5)	1 (12.5)
Pigeon	4	2 (50)	2 (50)
Total	197	39 (19.8)	31 (15.7)

Detection of Cryptosporidium species in poultry

C. baileyi, C. meleagridis, and C. parvum were identified through sequencing; however, C. baileyi was the most predominant species. Of the 31 positive samples, 17 were for C. baileyi (8.7%), 12 for C. meleagridis (6.0%), and 2 for C. parvum (1.0%). Cryptosporidium baileyi was detected in 11.9% (11/93) of sonalis, 5% (4/80) of broilers, 8.4% (1/12) of layers, and 25% (1/4) of pigeons. Cryptosporidium meleagridis was also detected in this study in 5.4% of sonalis, 5% of broilers, 8.4% of layers, 12.5% of natives, and 25% of pigeons. Moreover, Cryptosporidium parvum was identified in 1% of sonali and 1.3% of broiler (Table 2). Significant statistical differences were not found in relation to the different species of poultry and the relationship between age and infection (Table S2).

Table 2

Detection of Cryptosporidium spp. in poultry.

Poultry spp.	No. samples	No. positive (%)			Total positive (%)	95% CIa
		C. baileyi	C. meleagridis	C. parvum
Chicken (sonalis)	93	11 (11.9)	5 (5.4)	1 (1)	17 (18.3)	11.8–27.4
Chicken (broilers)	80	4 (5)	4 (5)	1 (1.3)	9 (11.3)	6.03–20.02
Chicken (layers)	12	1 (8.4)	1 (8.4)	0 (0)	2 (16.7)	4.7–44.8
Chicken (natives)	8	0 (0)	1 (12.5)	0 (0)	1 (12.5)	2.3–47.1
Pigeons	4	1 (25)	1 (25)	0 (0)	2 (50)	15–85
Total	197	17 (8.7)	12 (6.0)	2 (1.0)	31 (15.7)	11.4–21.5

Confidence interval.

Prevalence of Cryptosporidium spp. in different live bird markets (LBM)

Cryptosporidium spp. were found in most of the live bird markets in different areas of Dhaka, Bangladesh. The prevalence of Cryptosporidium spp. was high among chickens (10%–40%) and pigeons (50%) in Mirpur areas where LBM 4, 10, 12, and 13 were located and where C. baileyi, C. meleagridis, and C. parvum were detected. However, we found no Cryptosporidium in the areas of Kollyanpur, Mirpur new society, where LBM 6 and 11 were located (Table S3).

Sequence and phylogenetic analyses

All PCR-positive specimens were successfully sequenced and analysis of the nucleotide sequences of the 18S rRNA genes revealed the presence of three Cryptosporidium species: C. baileyi, C. meleagridis, and C. parvum. Direct sequencing of the 18S rRNA gene amplicons identified C. baileyi (8.7%; 17/197), C. meleagridis (6.0%; 12/197), and C. parvum (1.0%; 2/197). The sequences from C. baileyi, C. meleagridis, and C. parvum had 100% genetic similarity with sequences previously published in GenBank (JX548294, KT151550, KU744845, MK311146 HQ917077, KY448456, KY352486, MH062745, MF671870, MK491508 and LC270282).

ML analysis of 18S rRNA gene sequences revealed three distinct clusters among the isolates of cryptosporidia from poultry in the present study. Seventeen isolates clustered with C. baileyi, sharing 100% sequence identity with accession numbers (MN133967-MN133971, MN133976- MN133977, MN133980, MN133981, MN133983, MN133986, MN133987, MN133989- MN133991, MN133993 and MN133995). Two isolates clustered with C. parvum sharing 100% sequence identity with accession numbers (MN133979 and MN133984). The remaining 12 isolates belonged to C. meleagridis, sharing 100% sequence identity with accession numbers (MN133966, MN133972- MN133975, MN133978, MN133982, MN133985, MN133988, MN133992, MN133994 and MN133996) (Fig. 2). All 12 C. meleagridis-positive specimens generated the expected GP60 PCR product. However, only 11 of the 12 isolates were successfully sequenced. Nucleotide sequence analysis of the GP60 gene revealed that all of the subtypes belonged to the most common subtype, the IIIb family of C. meleagridis. We detected two novel subtypes, IIIbA21G2R1, in sonali chickens (n = 4) and a broiler chicken (n = 1) and IIIbA20G2R1, in a layer chicken (n = 1), that had a different nucleotide sequence within the trinucleotide repeat region. We also identified two other subtypes (IIIbA21G1R1 and IIIbA23G1R1) found in broiler chickens (n = 2), a native chicken (n = 1), a sonali chicken (n = 1) and a pigeon (n = 1). The GP60 gene of C. parvum was also amplified by specific sets of primers to reveal two subtypes (IIaA11G2R1 and IIaA13G2R1) identified in a sonali chicken (n = 1) and a broiler chicken (n = 1) that were previously reported in calf (Fig. 3). Therefore, these subtypes were named based on the established GP60 nomenclature [38,39].

Fig. 2

Phylogenetic tree based on partial sequences of the 18S rRNA genes for Cryptosporidium spp. A Phylogenetic tree was constructed without nucleotide gaps using the Maximum Likelihood analysis with 1000 replicates based on the T92 + I model [40]. Species, host, region of identification, and GenBank accession number are included. Newly obtained sequences are bolded. The Plasmodium falciparum sequence was used as an out-group. Only bootstrap values >50% from 1000 pseudo-replicates are shown. Evolutionary analyses were conducted in MEGA7 [21].

Fig. 3

Phylogenetic tree based on partial sequences of the GP60 genes for Cryptosporidium spp. Phylogenetic tree was constructed without nucleotide gaps using the Maximum Likelihood analysis with 1000 replicates based on the Tamura-Nei model [41]. Subtypes, host, region of identification, and GenBank accession number are included. Newly obtained sequences are bolded. C. meleagridis and C. parvum were subtyped in this study by use of reference sequences and observations. Only bootstrap values >50% from 1000 pseudo-replicates are shown. Evolutionary analyses were conducted in MEGA7 [21].

Discussion

Few studies have genotyped and subtyped Cryptosporidium in poultry around the globe. This study represents, 19.7% of Cryptosporidium prevalence in different breeds of poultry based on microscopic results. In contrast, a report mentioned high frequency (about 37%) of Cryptosporidium in broiler flocks in Morocco by using microscopy [18]; however, in China, lower Cryptosporidium infection rates of 3.4% in broilers and 10.6% in layer chickens were identified from fecal samples by using bright-field microscopy [44]. Furthermore, only 0.5% of chickens at poultry slaughterhouses in Iran were reported to be Cryptosporidium-positive [13]. The quantity of oocysts was not evaluated in this study. However, in a positive sample, the sensitivity of the primer that we used, there should be at least 101 oocysts found under microscope [17].

In this study, the positive rates of Cryptosporidium obtained by using molecular techniques were comparatively higher than those obtained by other investigators, such as 7.03% found in Germany [14] and 10% found in China [43,45], although the rate of 14.8% found in Brazil is similar to our findings [6]. In general, molecular technique is more sensitive than microscopy to detect infection. However, sometimes, microscopy showed high detection rate. It might be due to presence of PCR inhibitors in feces including bilirubin, bile salts, and complex polysaccharides, and thus PCR can be inhibited [28].

In the present study, Cryptosporidium baileyi was detected in sonali, broiler, and layer chickens and also in pigeons. These findings are supported by the work of Baroudi et al., [3], who reported a similar prevalence (5.5%; 5/90) in broiler chickens in Algeria using molecular techniques. Moreover, similar findings have been reported in Germany in broilers (5.7%; 9/158) and layers (8.3%; 1/12) [14]. Broiler chickens might act as sources of infection due to shedding of oocysts and may be responsible for transmission and infection [35]. Cryptosporidium baileyi is considered the most common avian species of Cryptosporidium worldwide and has a wide host range [29,33]. In the present study, C. baileyi was the predominant species and was identified in all groups of chickens, although C. meleagridis was also observed. C. baileyi was detected in sonali, broiler, and layer chickens and in pigeons in this study, whereas C. baileyi has been reported frequently in chickens and pigeons worldwide [16,25]. Cryptosporidium meleagridis was also detected in sonali, broiler, layer, and native chickens as well as in pigeons. Similarly, C. meleagridis was previously reported in 3.2% of chickens [25], 9% of broilers in Algeria [22], 5.3% of broilers in China [43,45] and 10% of layer chickens in China [44]; however, some reports have shown infection rates of C. meleagridis as high as 28.9% in chickens [3]. C. baileyi was found in all age groups of chickens, whereas, C. meleagridis was identified in 31 to 120-day-old layer chickens [44]. Both C. baileyi and C. meleagridis were found in >4 months of age of chicken [25]. The breeds of poultry in this study has different life span and genetic variation. We considered that young poultry were the risk group since their immune system is not developed. However, in the present study the infection of poultry was in adult group which might be due to stress factors for meat and egg production in poultry [14].

We identified two novel subtypes of C. meleagridis (IIIbA21G2R1 and IIIbA20G2R1) in sonali chickens, in one broiler chicken and in one layer chicken. The remaining 2 subtypes, IIIbA21G1R1 and IIIbA23G1R1, were identified in chickens and pigeons, consistent with previous reports in humans and birds [1,10,38]. The presence of C. meleagridis in poultry in live bird markets in this study raises questions regarding potential zoonotic transmission from poultry to humans. C. meleagridis is a public health concern in that it has also been found in humans, associated with gastrointestinal symptoms in both immunocompetent and immunocompromised individuals and mammals [5]. In Peru, two subtypes of C. meleagridis identified from AIDS patients were shared by birds (chicken, pigeon, or duck) in the same location [43,45]. The subtype IIIbA22G1R1 was detected in people from Sweden who had traveled to India or Thailand prior to infection [38]. These differences in prevalence might be related to the different breeds of poultry has different susceptibility and immunity due to genetic variation, and the situation of location of LBM, and the improper hygienic condition in poultry cages of LBM. Environmental factors and differences in host species may also be responsible. Unhygienic conditions in cages, overpopulation, and keeping different birds together have contributed to the high infection rates of Cryptosporidium [4].

C. parvum also has zoonotic potential and is sporadically found in birds [29]. In Germany, the most frequent species found in chickens and turkeys was C. parvum [14], which raises the possibility that poultry could act as a source of infection and mechanical vector for other zoonotic Cryptosporidium, besides C. meleagridis. However, in our study, the sanitary conditions were characterized as poor due to the lack of periodic cage cleaning and overpopulation. Also, the presence of ruminants in LBMs, which could be infected with C. parvum, could have caused the spread of oocysts in the surroundings, favoring ingestion by poultry. In contrast, migratory birds would act as a source of transmission, mechanical passage of oocysts, and contamination of the environment, even though they have low-level infections [26].

Avian species have been identified as the natural reservoir for C. meleagridis, and zoonotic transmission has been described from domesticated chickens to humans [35]. In urban areas of Bangladesh, C. meleagridis was the second most frequently identified species occurring in 13%, and also C. parvum was identified in 2% infecting children without diarrhea. However, in rural areas, the predominant species was C. meleagridis (90%) while C. parvum was much less prevalent (4%) causing subclinical cryptosporidiosis [37]. C. meleagridis and C. parvum have been identified in infants in Mirpur, Bangladesh. This might be anthropozoonotic; transmitted from chickens kept in households [20]. Interestingly, our study also detected C. meleagridis and C. parvum in LBMs in Mirpur areas. It is not possible to say whether the C. meleagridis subtypes identified in this study are related to subtypes that have infected humans due to lack of identified GP60 gene subtyping data of C. meleagridis in humans in Bangladesh. The C. parvum subtype IIcA5G3R2 and subtype IImA7G1 were identified in children with diarrhea aged <2 and 5 years, respectively, in Bangladesh. This subtype family has not been identified in any animal thus far and is generally considered “anthroponotic” [15,20]. In the present study, we detected two subtypes (IIaA11G2R1 and IIaA13G2R1) of C. parvum in broiler and sonali chickens that were previously reported as calf subtypes [46]. In the present study, Cryptosporidium was detected in pigeons; however, a detection rate of 7% was found in pigeons in Brazil and 25% in pigeons in Thailand [19,30]. Pigeons could be infected by contact with animals or their owners could passively spread oocysts in the surroundings. There is also a risk of the owners becoming infected due to contact with oocysts discharged by the pigeons [30]. The presence of C. meleagridis in domestic pigeons in China might lead to zoonotic transmission, especially to their handlers and the environment due to mice spreading the oocysts in water and food [23]. Market workers and customers were handling poultry in the markets could contaminate water, feed, and/or litter in poultry houses with oocysts of mammalian/human origin.

Conclusion

In conclusion, our research revealed that Cryptosporidium parasites are common among the live bird markets in Bangladesh. It is, therefore essential to pay attention to this pathogen. People and other animals could be infected with C. meleagridis and C. parvum via potential zoonotic transmission from poultry carrying the pathogen. Therefore, it is important to consider Cryptosporidium as a threat to public health as well as the economy.

Authors' contributions

MHBK and KK conceived of the presented idea. MHBK wrote the manuscript with input from all authors. MHBK carried out the experiments and analyzed the data. MHBK, FM, SL, and ABN organized and sort out all data. FM, YH, SL, FR and ABN contributed to the interpretation of the results.  XX and KK to investigate and supervised the findings of this work. All authors provided critical feedback, discussed the results and contributed to the final manuscript.

Declaration of Competing Interest

The authors have no conflicts of interest to declare.