Giardia genotyping

Debenham, John J., Tysnes, Kristoffer, Khunger, Sandhya & Robertson, Lucy J., 2017, Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesus macaques (Macaca mulatta) living in urban and semi-rural North-West India, International Journal for Parasitology: Parasites and Wildlife 6 (1), pp. 29-34 : 31-32

3.2. Giardia genotyping

Of the twenty-six Giardia positive samples selected for molecular characterisation, seventeen tested positive at one or more gene, with the SSU rRNA loci being the most sensitive ( Table 1).

# Cysts a DAPI b TPI c GDH d GDH e BG f SSU g

1 950 800 — B (KX787059) B (KX787059) B (KX787068) B (KX787044)

2 1150 600 — — — — B (KX787042)

3 200 150 — — B (KX787061) — B (KX787047)

4 130 70 — — — — Positive

5 190 60 — B (KX787060) — B (KX787069) —

6 110 50 — — — — B (KX787043)

7 110 50 — — — B (KX787055) B (KX787046)

8 80 50 — — — B (KX787056) B (KX787050)

9 50 40 — — — — B (KX787045) 10 70 20 — — — — B (KX787049) 11 40 20 — — — — —

12 30 20 — — — — Positive

13 30 20 B (KX787057) — — — —

14 20 20 — — — — Positive

15 20 20 B (KX787058) — — — B (KX787048) 16 160 10 — — — — —

17 80 10 — — — — Positive

18 40 10 — — — — —

19 320 0 — — — — Positive

20 240 0 — — — — —

21 170 0 — — — — —

22 160 0 — — — — —

23 130 0 — — — — —

24 130 0 — — — — —

25 130 0 — — — — —

26 110 0 — — — — Positive

TPI, triosephosphate isomerase; GDH, glutamate dehydrogenase; BG, beta giardin; SSU, small subunit rRNA; -, PCR negative; Positive, amplification on PCR however no sequencing results; Assemblage (Accession number) provided where sequence of PCR products was obtained.

a Number of Giardia cysts used for DNA isolation.

b Number of DAPI positive Giardia cysts used for DNA isolation.

c Sulaiman et al. (2003).

d Caccio et al. (2008).

e Read et al. (2004) & Robertson et al. (2006).

f Lalle et al. (2005).

g Hopkins et al. (1997) & Read et al. (2002).

Amplification by PCR was more likely if more than twenty DAPIpositive cysts were used for DNA isolation, 80% (12/15), than if 10 or less DAPI positive cysts were used, 27% (3/11) (p <0.05). There was no observed correlation observed between the total number of cysts and the likelihood of a sample being positive by PCR.

Sequencing of PCR products revealed Assemblage B in all samples. Sequences were submitted to GenBank and Accession numbers are provided ( Table 1). Multiple alignment of consensus sequences at the TPI, GDH, BG and SSU rRNA genes showed Giardia excreted by macaques to be very similar to each other, 98 — 99%, with differences primarily due to ambiguous nucleotides. Importantly, there was heterozygosity of alleles within the sequences corresponding to the reverse internal primer at the BG gene and the reverse internal primer at the SSU rRNA genes. BLAST results of macaque sequences at the TPI, GDH and BG genes showed 99% identity to Giardia isolates from humans, common marmosets and a beaver. Two samples, 5 and 8 ( Table 1), showed 100% identity at the BG gene to a Giardia isolate from a sheep and human.

3.3. Prevalence of Cryptosporidium spp . oocysts shed by wild rhesus macaques

Examination of rhesus macaque faecal samples using immunofluorescent microscopy revealed the presence of Cryptosporidium oocysts in 1 of 170 samples, with this animal from Troop 3. This sample contained 50 oocysts per gramme of faeces, all of which stained positively with DAPI, however was negative by PCR at the SSU rRNA gene.

3.4. Entamoeba coli and unknown Entamoeba spp . in wild rhesus macaques

Examination of rhesus macaque faecal samples using a genus-specific conventional PCR revealed the presence of Entamoeba spp . in 79% (132/168) of samples. There was no significant difference in the prevalence of Entamoeba spp . between Troops 1, 2, 3 and 4; 78% (43/55), 69% (31/45), 83% (19/23) and 87% (39/45) respectively (p = 0.21).

Multiplex PCR for E. histolytica , E. dispar and E. moshkovskii , did not result in amplification in any of the samples (0/168). Species-specific PCR for E. coli resulted in amplification in 49% (63/128) of samples positive at the genus-specific PCR. Thus, in the other 51% (65/128), no species of Entamoeba was identified. There was a significant difference in the prevalence of E. coli between Troops 1, 2, 3 and 4; 26% (11/42), 75% (21/28), 56% (10/18) and 45% (21/39), respectively (p <0.01).

3.5. Giardia and Cryptosporidium in calves living in association with wild rhesus macaques (Troop 3)

Examination of faeces from domestic calves living together with Troop 3, revealed Giardia spp. cysts in 64% (9/14) of samples. Calves excreted 55 to 19 250 cysts per gramme faeces (mean, 4746; median, 302). Five positive samples were analysed further by PCR, and all five tested positive at one or more loci. Sequencing of PCR products revealed Assemblage A (KX787052, KX787054) in two calves, Assemblage A1 (KX787067) in one calf, Assemblage E (KX787051, KX787063, KX787065) in one calf, and a mixed infection of Assemblage A1 (KX787062, KX787053) and E (KX787064, KX787066) in one calf.

Cryptosporidium spp . oocysts were detected in 36% (5/14) of samples. Calves were excreting 100 to 5000 oocysts per gramme faeces (mean; 1480, median; 700). PCR at the SSU rRNA gene was negative for all 5 samples.