The protozoan is currently considered as a complex of species, due to the existing genetic differences among isolates infecting different hosts [
Substantial differences found in glutamate dehydrogenase (
Cattle, pigs, and sheep are susceptible to infections with species-specific assemblage E and also with zoonotic infections of
For some time, most studies on genotypic characterization were only conducted with a single gene locus; however, studies have shown inconsistent results when distinct loci are sequenced. Thus, the genotyping of more than one gene may improve the attribution of the assemblage to its respective isolate, helping in the understanding of the epidemiology of giardiasis [
This work has the purpose of assessing the genotypic variability among isolates of
This work has been approved by “Comitê de Ética na Utilização de Animais da Universidade Federal de Uberlândia” (CEUA-UFU) (Ethics Committee on Animal Use of the Federal University of Uberlândia), protocol 003/12.
Cattle, pigs, and sheep, with an age range of 0 to 10 months, both males and females, and with different breeds from the microregion of Uberlândia, Minas Gerais state, southeastern region of Brazil have been included in the study.
Cattle were distributed in 17 farms; 5 of them were Holstein cattle (PO) (for milk production) and 12 with Girolando cattle, were milk and beef production. Sheep came from a single farm with Corriedale breed with meat and wool production, and pigs came from 10 commercial farms, with Landrace breed.
Feces were collected individually straight from the rectal ampulla of cattle and sheep. Regarding pigs, samples were collected in pools, straight from the floor of the stalls, as they were grouped in lots of 30 to 40 animals, according to their age. Each pool was considered as a sample.
Due to the intermittent pattern of elimination of
Feces were collected and stored in plastic bags identified with the number of each animal or lot, the name of the farm, and the date of collection and were sent to Laboratório de Parasitologia (laboratory of parasitology) of Universidade Federal de Uberlândia (UFU) for processing.
Samples were considered positive for
After resuspension in 500
In order to amplify the fragments of glutamate dehydrogenase (
The fragment of 530 base pairs of
In order to amplify the fragment of
PCR and nested-PCR reactions were performed in Mastercycler pro thermocycler (Eppendorf, Brazil) according to the protocols described by Cacciò et al. [
Bands of interest were visualized through the agarose gel electrophoresis technique at 2% (P/V), and stained with ethidium bromide at 0.5
Positive nested-PCR products were purified with Sephacryl 400 resin (Ilustra-MicroSpin S400 HR Columns) and sequenced in a single direction. Reactions were performed in a Mastercycler pro thermocycler (Eppendorf, Brazil) using Big Dye terminator V.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Products were read using the automatic sequencer ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
The quality of the partial sequences and the clustering of the fragments were obtained with the use of Sequence Scanner version 1.0 (Copyright Applied Biosystems, Foster City, CA, USA). Nucleotide alignment was performed manually using BioEdit Sequence Alignment Editor (Hall, 1999) and using as a base the homologous sequences available on GenBank: M84604 (assemblage A), AY826193 (assemblage B), U60982 (assemblage C), U60986 (assemblage D), AY178741 (assemblage E), and AF069057 (assemblage F) for
In order to determine the phylogenetic relationship among assemblages, phylograms were constructed using Mega v.5.1 Beta, with the neighbor-joining method, with bootstrap values established in 1000 replicates.
Two hundred and fifty-six fecal samples from cattle, 105 from sheep, and 90 from pigs were collected. Due to the fecal collection in triplicate, 768 and 315 fecal examinations were performed in cattle and sheep, respectively.
Positivity for
Out of all positive samples (
Out of 25 samples with amplified fragments of
In this study, 44 sequences were obtained and analyzed, 24 from
Fourteen isolates from sheep and nine from cattle, in which
When analyzing each sequence individually and comparing them to their respective base sequence, an intra-assemblage variation was seen in three isolates of sheep identified as E by the
Variation of
Sample |
Positions | |||
---|---|---|---|---|
520 | 585 | 630 | 654 | |
AY178741* | C | G | C | A |
LaNUdi010 | T | A | T | G |
LaNUdi012 | T | A | T | G |
LaNUdi013 | · | A | T | G |
*Base sequence of assemblage E stored at GenBank.
The two isolates of pigs differed by genotyping; one was identified as assemblage E and the other as D. The isolate identified as assemblage D was heterogeneous, with double peaks throughout the gene (Figure
Overlapped nucleotides in chromatogram of a pig sample identified as assemblage D by the sequencing of
The phylogenetic relationship among isolates genotyped by sequencing
Phylogenetic relationships of
Genotyping of
Two isolates of sheep and three of cattle had polymorphic sites along the gene sequence between assemblages BIII and E, where the colocalization of conserved nucleotides was observed (Table
Overlapped nucleotides in isolates from cattle and sheep characterized as E and BIII genotyping by
Isolates | |||||||
---|---|---|---|---|---|---|---|
AF069561* | AY228645* | LaNUdi001** | LaNUdi007*** | CaNUdi003** | CaNUdi006** | CaNUdi008** | |
Nucleotides positions and replacements | |||||||
347 | C | T | T | · | · | · | · |
350 | C | T | A | · | · | · | · |
374 | G | A | A | · | · | · | · |
396 | C | T | T | T | · | · | · |
397 | T | C | C | · | · | · | · |
398 | T | C | C | · | · | · | · |
405 | C | T | · | T | · | · | · |
408 | T | A | A | · | · | · | · |
411 | T | A | A | · | · | · | · |
426 | G | A | A | A | · | · | · |
429 | T | C | C | C | · | · | · |
432 | T | G | G | · | · | · | · |
439 | T | T | C | · | · | · | · |
440 | G | A | A | · | · | · | · |
442 | C | T | T | · | · | · | · |
462 | C | T | T | · | · | · | · |
468 | T | C | C | · | · | · | · |
471 | G | A | A | · | · | · | · |
473 | A | G | G | · | · | · | · |
483 | T | A | A | · | · | · | · |
495 | C | T | T | · | · | · | · |
501 | A | G | · | · | A | A | A |
504 | C | T | T | · | · | · | · |
507 | C | T | T | · | · | C | · |
518 | T | A | A | · | · | · | · |
528 | G | C | · | · | T | T | · |
529 | C | T | T | · | · | · | · |
532 | A | A | A | · | A | A | · |
533 | G | A | · | · | · | G | · |
534 | C | G | · | · | C | C | · |
535 | C | T | · | · | · | C | C |
537 | T | C | · | · | T | T | T |
540 | A | G | · | · | A | A | · |
548 | G | G | · | · | A | A | · |
549 | A | G | G | · | · | · | · |
552 | C | T | T | · | · | C | · |
561 | A | G | · | · | A | A | · |
583 | A | G | · | · | A | · | · |
585 | T | C | · | · | T | · | · |
597 | T | C | C | · | · | · | · |
606 | G | T | T | · | · | · | · |
618 | G | A | · | · | G | G | G |
624 | A | G | · | · | · | A | · |
625 | G | G | · | · | · | · | A |
627 | T | G | · | · | T | T | T |
633 | T | G | G | · | · | · | · |
642 | C | T | · | · | · | C | · |
645 | C | T | · | · | C | C | · |
647 | C | T | G | · | · | · | · |
693 | A | A | · | G | · | · | · |
*AF069561: assemblage BIII; *AY228645: assemblage E; **isolates identified as assemblage BIII, ***isolates identified as assemblage E; dots indicate identity to the reference sequences.
When comparing sequences from sheep obtained in this study to those used as reference, four out of ten isolates were homologous to the reference sequence, with one being homologous to the sequence number JQ837919.1 and the others being homologous to the sequence number JQ837808.1, both stored in the Genbank. Out of the six remaining isolates of sheep, each one was different, with no homology to any sequence described, being considered new, and inserted into the Genbank under the numbers KC85814 to KC858149.
Among the sequences from cattle, one isolate was homologous to the other, present in the Genbank (JQ837925.1). The other isolates from cattle differed among each other and are present in the GenBank under the numbers KC858151 to KC858157.
The isolate from pigs was not identical to any other described ones, thus being considered new and inserted into the Genbank under the number KC858150.
While constructing a phylogenetic tree (Figure
Phylogenetic relationships of
Out of all the samples (
Genotyping and subgenotyping of samples from sheep, pigs, and cattle from the sequencing of
Samples |
|
|
|
---|---|---|---|
Assemblage | Assemblage | Subassemblage | |
LaNUdi001 | E | E | |
LaNUdi002 | E | — | |
LaNUdi003 | E | A | II |
LaNUdi004 | E | — | |
LaNUdi005 | E | E | |
LaNUdi006 | E | E | |
LaNUdi007 | E | B | III |
LaNUdi008 | E | — | |
LaNUdi009 | E | — | |
LaNUdi010 | E | E | |
LaNUdi011 | E | B | III |
LaNUdi012 | E | — | |
LaNUdi013 | E | — | |
LaNUdi014 | E | A | II |
LaNUdi015 | — | E | |
LaNUdi016 | — | E | |
PiNUdi002 | E | — | |
CaNUdi001 | E | E | |
CaNUdi002 | E | E | |
CaNUdi003 | — | E | |
CaNUdi005 | — | E | |
CaNUdi006 | E | E | |
CaNUdi008 | — | E | |
CaNUdi010 | E | E | |
CaNUdi012 | E | — | |
CaNUdi013 | E | — | |
CaNUdi014 | E | — | |
CaNUdi016 | E | — | |
CaNUdi018 | — | E | |
CaNUdi022 | — | B | III |
La: lambs, Ca: cattle, Pi: pig.
The region studied is very important in the livestock production in Brazil, with 3% of the total amount of heads of cattle in the country. Nevertheless, there are no studies published on the presence and epidemiological characteristics of
PCR for both genes has failed to amplify some positive samples with the microscopic technique in this study. The failure may be attributed to the following: the presence of PCR inhibitors in the feces, the small amount of cysts, the small amount of target DNA present in the samples, and the association of these factors with the low efficiency of the DNA extraction process [
Although authors such as Leonhard et al. [
According to Read et al. [
In the present study, it was observed that when using
The sequencing of the
When genotyping
Heterogeneous sequences have been observed when genotyping
Studies have reported that 15% of isolates genotyped have genotypic inconsistencies between two markers [
Genetics of the
The authors would like to thank the farm owners who authorized them to collect fecal material and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais” (FAPEMIG) for the financial support.