Occurrence and multilocus genotyping of Giardia duodenalis from post-weaned dairy calves in Sichuan province, China

Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3–12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E (n = 115, 91.3%), A (n = 3, 2.4%), and A mixed with E (n = 8, 6.3%) were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China.


Introduction
Giardia is one of the most common parasitic protists that infects both humans and animals, poses a considerable threat to human and animal health globally [1,2]. Among the six species of Giardia, only Giardia duodenalis can infect humans and animals (domestic, farmed and wild animals) [3,4]. The life cycle of Giardia is relatively simple; involving two developmental stages of rapid multiplying trophozoites and infectious cysts, transmitted via the faecal-oral route (i.e., faeces, contaminated water or food) [1,5]. Humans and animals infected with Giardia usually show symptoms such as diarrhoea, abdominal cramps, weight loss, malabsorption or recessive infections without obvious clinical symptoms [5,6]. Young animals are more a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 susceptible to giardiasis than adults and likely linked to the immature immune status, which lead to substantial production losses to the livestock industry [6].
Giardia duodenalis is recognised as a complex comprised of at least eight different assemblages (A-H) [4,7]. Assemblage A and B can infect various mammals including humans, and are considered as the zoonotic assemblages. The other assemblages are either host specific or have narrow host ranges [6]. Cattle are dominantly infected with G. duodenalis assemblage E. Although there are fewer reports of zoonotic assemblages A and B, cattle are recognized as the contributor of the zoonotic sources of infection [6]. Many recent studies have focused on the infection of G. duodenalis in dairy calves, and the occurrence has been found to be significantly different between pre-and post-weaned stages [8][9][10][11][12][13]. In China, studies have also revealed different infection rates in pre-and post-weaned dairy calves, e.g., in Liaoning [14], Xinjiang [15], Hubei [16] and Guangdong [17] provinces. In our previous study, we conducted a preliminary study on G. duodenalis infection in pre-weaned calves in Sichuan province, China [18]. However, information regarding the occurrence in post-weaned dairy calves in Sichuan province is limited.
In this study, we further investigated the occurrence and genetic diversity of G. duodenalis in post-weaned calves from Sichuan province by using multilocus genotype (MLG) data and by assessing the zoonotic potential.
This study was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Sichuan Agricultural University (DYY-S20174604). Permission was obtained from the farm owners before collecting the fecal samples.

DNA extraction
Before DNA extraction, stored faeces were washed with distilled water to remove the potassium dichromate. Genomic DNA was extracted from~250 mg of the individual samples using the PowerSoil DNA isolation kit (MOBIO, USA), according to the manufacturer's instructions. All DNA samples were stored at −20˚C prior to analysis by Giardia PCR.

PCR amplification
Giardia duodenalis DNA was detected by nested PCR amplification of the beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. The primers and amplification conditions used in this study have been described previously [19]. Positive and negative controls were included in each test. The secondary PCR products were visualized under UV light after electrophoresis on 1% agarose gel mixed with Golden View.

Sequence analysis
All positive secondary PCR products were sent to BGI Tech Solutions (Liuhe Beijing) Co., Limited and were sequenced in both directions. Sequences were aligned with reference sequences from GenBank using BLAST (http://blast.ncbi.nlm.nih.gov) and Clustal X (http:// www.clustal.org/). Specimens that were successfully subtyped at all three loci were used to investigate the MLGs of G. duodenalis. Sequences were concatenated for each positive isolate to form a multilocus sequence in accordance with (bg + tpi + gdh). All the concatenated MLGs were used in a neighbour-joining analysis, with the Kimura-2 parameter model calculated using the Molecular Evolutionary Genetics Analysis (MEGA) version 7 (http://www.megasoftware.net/). The genetic pedigree of the assemblage E MLGs in Sichuan was assessed by using eBURST 3.0 (http://eBURST.mlst.net).
The novel G. duodenalis genotypes obtained at bg, tpi and gdh loci in this study were deposited in GenBank under the accession numbers: MK642904-MK642913.

Statistical analysis
The variation in G. intestinalis prevalence among the different regions was analysed by χ2 test using SPSS Statistics version 20.0. Differences were considered significant at P < 0.05.
Numerous studies have reported that the prevalence of G. duodenalis is inversely associated with animal age [1,8,11]. However, we measured a significantly higher occurrence of G. duodenalis in post-weaned calves (41.2%) compared with previous measurements of pre-weaned calves (26/278, 9.4%) [18] (P < 0.01, X 2 = 76.623, df = 1); which was similar to some reports from China [15,16] and the USA [12,13]. Moreover, we analysed the infection rates between intensive feeding and free-ranging farms and found consistent results with our previous study [18], i.e., no significant differences between the two breeding systems (P = 0.179, X 2 = 1.809, df = 1). Similarly, a study conducted in Malaysia also showed no significant differences in the occurrence of G. duodenalis infection between intensive and semi-intensive farms [8].
Of the 126 G. duodenalis-positive specimens, 121 were positive in bg gene, 101 in tpi gene, and 111 in gdh gene. Giardia duodenalis assemblage E (n = 115, 91.3%), assemblage A (n = 3, 2.4%), and mixed assemblage (A and E) (n = 8, 6.3%) were identified among the 126 genotyped specimens (Table 1), which is consistent with other studies conducted in China [14], the USA [11] and India [10]. All of the assemblage A isolates identified in the present study belonged to subtype A1, which has mostly been detected in animals [1,5]. However, there have been a few reports of human infections of subtype A1, e.g., in China [23], Portugal [24], Mexico [25] and Brazil [26], which suggests that dairy calves may potentially play a role in the zoonotic transmission of G. duodenalis infection from cattle to humans. Sequence analyses revealed 12, 9 and 15 subtypes identified at the bg, tpi and gdh loci, respectively. Of the bg subtypes, eight had previously been identified. The remaining four sequences represented subtypes E17-E20 (MK642904-MK642907) that were previously unpublished. Of the tpi subtypes, eight subtypes identified before and the remaining one sequence subtype E25 (MK642913) was previously unpublished. Of the gdh subtypes, ten were known and five were previously unpublished: E21-E25 (MK642908-MK642912). Of these subtypes, the most common were E9 (n = 20, bg subtype), E3 (n = 64, tpi subtype) and E10 (n = 48, gdh subtype). The high genetic diversity of assemblage E in this study was consistent with previous studies [18,27,28], which may be related to intra-assemblage genetic recombination [19,29].
Furthermore, MLG analysis of the bg, tpi and gdh genes was used to systematically characterize intra-assemblage genetic diversity and to determine the genotype of G. duodenalis. A total of 94 specimens were successfully sequenced at the bg, tpi and gdh loci, which formed 34 assemblage E MLGs (Table 2), one assemblage A MLG and seven mixed assemblage (A and E) MLGs. The most common MLGs were the MLG E48 (n = 11) and MLG E80 (n = 11), followed by MLG E94 (n = 9) and MLG E3 (n = 6). Among them, the most widely distributed MLG was MLG E3, which was detected in four regions (Chengdu, Deyang, Meishan and Ya'an). To reveal the genetic relationship between MLGs, we constructed clonal pedigree maps of the 34 assemblage E MLGs in the present study and the 19 assemblage E MLGs in our previous study [18] using eBURST software. Two clonal complexes and five singletons were observed (S1 Fig). MLG E3 is the primary founder of clonal complex 1, which is consistent with the results found in Shanghai [28]. Clonal complex 2 was formed by three assemblages E: MLG E94-E96. MLG E69, MLG E78, MLG E83, MLG E87 and MLG E98 were singletons. To better understand the diversity between assemblage E MLGs in Sichuan and in the other provinces of China (Henan [30], Gansu [31], Ningxia [31], Shaanxi [20], Xinjiang [15], Shanghai [28] and Guangdong [17,27]), a phylogenetic evolutionary tree was constructed. The phylogenetic tree (S2 Fig) showed that MLG E3 has also been found in Gansu [31], Xinjiang [15], Shanghai [28] and Guangdong [27], which indicates the wide distribution of this MLG subtype in China. However, the predominant subtype needs further elucidation. As seen from the phylogenetic tree, the 218 assemblage E MLGs identified in China were highly diverse and formed multiple evolutionary branches. The 53 assemblage E MLGs from Sichuan province showed a scattered distribution in the phylogenetic tree, which indicated that geographical segregation was not strict in our study.

Conclusion
The present study demonstrated a high occurrence of G. duodenalis in post-weaned calves in Sichuan province, China. Both assemblage E and zoonotic assemblage A were detected. MLG analysis revealed a high genetic diversity in assemblage E. MLG E3 was shown to be not only the primary MLG subtype in Sichuan province but also the most widely distributed MLG subtype throughout China. The phylogenetic tree was constructed using a concatenated dataset of bg, tpi and gdh gene sequences, bootstrap values greater than 50% from 1000 replicates are shown. Isolates from the present study are indicated by black triangles, and isolates from our previous study [18] are indicated by black circles.