Open Access

Molecular identification of zoonotic and livestock-specific Giardia-species in faecal samples of calves in Southern Germany

  • Julia Gillhuber1Email author,
  • Louise Pallant2,
  • Amanda Ash2,
  • RC Andrew Thompson2,
  • Kurt Pfister1 and
  • Miriam C Scheuerle1
Parasites & Vectors20136:346

https://doi.org/10.1186/1756-3305-6-346

Received: 21 October 2013

Accepted: 4 December 2013

Published: 10 December 2013

Abstract

Background

Giardia-infection in cattle is often subclinical or asymptomatic, but it can also cause diarrhoea. The livestock-specific species Giardia bovis is the most frequently observed in cattle, however, the two zoonotic species Giardia duodenalis and Giardia enterica have also been found. Therefore calves are thought to be of public health significance. The aim of this study was to obtain current data about the frequency of the different Giardia-species in calves in Southern Germany.

Findings

Faecal samples of calves (diarrhoeic and healthy) in Southern Germany, diagnosed Giardia-positive by microscopy, were characterised by multi-locus PCR and sequencing.

Of 152 microscopically Giardia-positive samples 110 (72.4%) were positive by PCR and successfully sequenced. G. bovis (Assemblage E) was detected in 101/110 (91.8%) PCR-positive samples, whilst G. duodenalis (Assemblage A) was detected in 8/110 (7.3%) samples and a mixed infection with G. duodenalis and G. bovis (Assemblage A+E) was identified in 1/110 (0.9%) samples. The sub-genotypes A1, E2 and E3 were identified with the β-giardin and the glutamate dehydrogenase genes. In the majority of diarrhoeic faecal samples a co-infection with Cryptosporidium spp. or Eimeria spp. was present, however, there were some in which G. bovis was the only protozoan pathogen found.

Conclusions

The results suggest that there is potentially a risk for animal handlers as calves in Southern Germany are, at a low percentage, infected with the zoonotic species G. duodenalis. In addition, it was found that G. bovis was the only pathogen identified in some samples of diarrhoeic calves, indicating that this parasite may be a contributing factor to diarrhoea in calves.

Keywords

PCRDiarrhoeaProtozoanGiardia assemblagesCattleGiardia duodenalis morphological group

Findings

Background

Worldwide the protozoan Giardia spp. is one of the most common intestinal parasites in humans (reviewed in [1, 2]) and also a frequent enteric parasite in animals including companion animals, livestock and wildlife [2]. According to Monis et al. [3] there are eleven species within the genus Giardia. Six of them, formally known as Assemblages A-G of the Giardia duodenalis morphological group, are genetically but not morphologically distinguishable. They can infect humans and mammals, with some being host specific and others having low host specificity.

Giardia- infection in cattle is often subclinical or asymptomatic, but this infection can also cause symptoms including acute or chronic diarrhoea, reduced weight gain and ill thrift in young calves [4, 5]. Although the prevalence of Giardia in cattle around the world varies considerably (reviewed in [5, 6]), longitudinal studies have shown cumulative infection rates in calves of 100% [7, 8]. The two zoonotic species G. duodenalis (Assemblage A) and G. enterica (Assemblage B) and the livestock-specific species G. bovis (Assemblage E) are able to infect cattle with G. bovis being found most frequently followed by G. duodenalis[913]. Therefore, calves are thought to be of public health significance both as a source of waterborne outbreaks of giardiasis in humans and as a risk to in-contact animal handlers [2, 14].

Current data on the occurrence of the different Giardia species in German calves is only available for 2–16 week-old calves from farms around Berlin. In that study (15) a commercially available monoclonal antibody-based ELISA was used and Giardia was detected in 100% of the farms and 51.2% of the animals sampled. Subsequent molecular characterisation ascertained G. bovis (Assemblage E) was the most common species present, but infections with G. duodenalis (Assemblage A) and mixed infections of G. duodenalis and G. bovis (Assemblage A+E) were also found [15].

Thus, the aim of this study was to obtain current data about the frequency of the different Giardia species in calves of a wider range of age in Southern Germany.

Methods

Samples

Faecal samples of calves from the southern federal states of Germany, Bavaria and Baden-Württemberg, were sent to the Diagnostic Laboratory of Comparative Tropical Medicine and Parasitology, LMU Munich, Germany for microscopy analysis. Giardia spp., Cryptosporidium spp. and Eimeria spp. were detected using the carbolfuchsin-stained direct faecal smear [16] and the merthiolate iodine formaldehyde concentration (MIFC) with the addition of Lugol’s solution [17]. Samples from 152 calves between 3 and 130 days of age (mean age: 50.7 days, n = 138) were diagnosed Giardia-positive by the MIFC-method between June 2011 and January 2013 and stored at −20°C. In February 2013 these samples were preserved in 70% ethanol and sent to the School of Veterinary and Life Sciences, Murdoch University, Australia, for molecular characterisation.

DNA extraction

DNA was extracted from faecal samples using the Maxwell® 16 Tissue DNA Purification Kit (Promega, Madison, USA) with the Maxwell® 16 Instrument (Promega). In addition to the recommended protocol, 1 μl of the final elution was further diluted by adding 4 μl of Water-ultra pure grade (Fisher Biotech Perth, Australia). Both neat and dilute templates were used in PCRs.

PCR amplification

For the amplification of the 18S rRNA gene and the β-giardin gene a nested PCR was carried out and for the amplification of the glutamate dehydrogenase (GDH) gene a semi-nested PCR was performed. Details of primers and cycling conditions are listed in Table 1.
Table 1

PCR conditions and primers

Target gene

Number of reaction

Length of amplification (bp)

Primer

Cycle condition

Reaction volume

Reference

18S rRNA

Primary reaction

292

Forward primer: RH11

a

Total volume 25 μl

[18]

5’-CATCCGGTCGATCCTGCC-3’

Reverse primer: RH4

96°C, 45 s

d

5’-AGTCGAACCCTGATTCTCCGCCAGG-3’

50°C, 30 s

0.15 μl Taq-Ti hot start DNA polymerasee

 

72°C, 45 s

→ 35 cycles

5% dimethyl sulfoxide (DMSO)f

b

Secondary reaction

130

Forward primer: GiarF

a

2 μl from the 1st-round PCR reaction

[19]

5’-GACGCTCTCCCCAAGGAC-3’

Reverse primer: GiarR

96°C, 45 s

5’-CTGCGTCACGCTGCTCG-3’

55°C, 30 s

 

72°C, 45 s

→ 35 cycles

b

β-giardin

Primary reaction

753

Forward primer: G7

a

Total volume 25 μl

[20]

5’-AAGCCCGACGACCTCACCCGCAGTGC-3’

Reverse primer: G759

95°C, 30 s

d

5’-GAGGCCGCCCTGGATCTTCGAGACGAC-3’

50°C, 30 s

0.15 μl Tth Plus DNA polymerasee

 

72°C, 60 s

 

→ 40 cycles

b

Secondary reaction

511

Forward primer: B-F

a

2 μl from the 1st-round PCR reaction

[21]

5’-GAACGAACGAGATCGAGGTCCG-3’

Reverse primer: B-R

96°C, 45 s

5’-CTCGACGAGCTTCGTGTT-3’

55°C, 30 s

 

72°C, 45 s

 

→ 35cycles

 

b

GDH

Primary reaction

not given

Forward primer: GDHeF

c

Total volume 25μl

[19]

5’-TCAACGTYAAYCGYGGYTTCCGT-3’

Reverse primer: GDHiR

94°C, 30 s

d

5’-GTTRTCCTTGCACATCTCC-3’

50°C, 30 s

0.2 μl Tth Plus DNA polymerasee

 

72°C, 60 s

 

→ 40 cycles

 

b

Secondary reaction

432

Forward primer: GDHiF

c

2 μl from the 1st-round PCR reaction

[19]

5’-CAGTACAACTCYGCTCTCGG-3’

Reverse primer: GDHiR

94°C, 30 s

5’-GTTRTCCTTGCACATCTCC-3’

60°C, 30 s

 

72°C, 60 s

 

→ 40 cycles

 

b

a: Initial activation step: 96°C, 5 min.

b: Final extension: 72°C, 7 min.

c: Initial activation step: 94°C, 5 min.

d: used substances: 2 μl diluted DNA template, 2.5 μl 10x Reaction Buffer , 2.5 μl MgCl2 (25 mM), 1 μl dNTPs (5 mM) (Promega), 1 μl of each primer (10 μM), Water-ultra pure grade (Fisher Biotech Perth, Australia).

e: Fisher Biotech Perth, Australia.

f: Sigma–Aldrich St. Louis, Missouri.

DNA sequencing

PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman coulter, Beverly, USA) as per the manufacturer’s instructions. Sequence reactions were performed using the Big Dye Terminator Version 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. PCR products were sequenced with the second round primers (1 μl [2.5 μM]). The cycling conditions for nucleotide sequencing are: 1 cycle of 96°C for 2 min and 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. Reactions were electrophoresed on an ABI 3730 48 capillary machine.

Species identification

Sequences were analysed using Sequencher 4.8 (Gene Codes, Ann Arbor, MI, USA) and compared to published sequences (Table 2) to identify species and sub-genotype information.
Table 2

GenBank accession numbers used for alignment with Giardia sequences

  18S rRNA

 

  β-giardin

 

   GDH

 

AI

AF199445

A1

X14185

A

DQ100288

AI

M54878

A2

AY545645

A

M84604

AII

AF199446

A2

FN386482

A1

DQ414242

AIII

AF199447

A5

AY545643

A2

L40510

B

U09491

A8

AY545649

B

AY826193

B

U09492

B

AY072728

B3

AF069059

C

AF199449

B

AY647266

B4

AY178750

D

AF199443

C

AY545646

C

U60982

E

AF199448

C

FJ009206

D

U60986

E

DQ157272

D

AY545648

E

AY178741

F

AF199444

E

EU189375

F

AF069057

G

AF199450

E1

AY072729

G

AF069060

  

E2

AY545650

  
  

E3

AY653159

  

Results

Of the 152 samples, diagnosed Giardia- positive by microscopy, 110 (72.4%) were positive by PCR and successfully sequenced.

Sequence analysis identified the presence of G. bovis (Assemblage E) in 101/110 (91.8%) PCR-positive samples, G. duodenalis (Assemblage A) in 8/110 (7.3%) samples and a mixed template of G. duodenalis and G. bovis (Assemblage A+E) in 1/110 (0.9%) samples. Using the β-giardin and GDH genes it was possible to identify sub-genotypes within the species G. bovis (E2 and E3) and G. duodenalis (A1) (Table 3).
Table 3

Genotypic characterisation of Giardia spp. isolates at different loci

18S rRNA

β-giardin

GDH

18S rRNA and β-giardin

18S and GDH

18S rRNA, β-giardin and GDH

A (5)

A1 (1)

A1 (1)

E, E (1)

E, A1 (1)

A, A1, A (1)

E (85)

E3 (1)

E (1)

E, E2 (1)

E, E (1)

E, E3, E (3)

   

E, E3 (8)

  

Of the 110 PCR-positive samples 94 (85.5%) samples amplified at one locus, whereas 12/110 (10.9%) and 4/110 (3.6%) samples amplified at 2 and 3 loci, respectively. 18S amplified most frequently (106/152 samples, 69.7%), whereas β-giardin and GDH amplified comparatively rarely (16/152, 10.5%; 8/152, 5.3%) (Table 3).

Table 4 shows that in the majority of the calves with diarrhoea a co-infection with Cryptosporidium spp. or Eimeria spp. was present.
Table 4

Distribution of mono- and mixed infections of Giardia -positive calves in relation to faecal consistency

  

Total

Monoinfection with Giardia spp.

Coinfection with Cryptosporidium spp.

Coinfection with Eimeria spp.

MIFC positive

Total

152

66

15

71

With diarrhoea

62

25

10

27

Without diarrhoea

90

41

5

44

PCR: G. duodenalis

Total

8

-

3

5

With diarrhoea

4

-

2

2

Without diarrhoea

4

-

1

3

PCR: G. bovis

Total

101

48

8

45

With diarrhoea

38

17

6

15

Without diarrhoea

63

31

2

30

PCR: G. duodenalis + G. bovis

Total

1

1

-

-

With diarrhoea

-

-

-

-

Without diarrhoea

1

1

-

-

Discussion

The results of this study reveal that the livestock-specific species G. bovis (Assemblage E) is the most frequent species (91.8%) in calves in Southern Germany. The zoonotic species G. duodenalis (Assemblage A) was found in a low number of samples (7.3%), while a mixed infection of G. duodenalis and G. bovis was identified in only one sample (0.9%). G. enterica (Assemblage B), the second zoonotic species, was not detected in this study.

Similarly in another study on German calves, the same species were detected and G. bovis was also found most frequently; however, there was a higher proportion of infection with G. duodenalis as well as with mixed infections than observed in this study [15].

Finding G. bovis in the majority of Giardia-infections in calves and G. duodenalis in only some cases also concurs with the results of former studies on cattle [1012, 2224]. In some studies G. bovis was the only species identified in calves [9, 25]. G. enterica was not detected in this study, which is in accordance with the results of many previous studies although several did find this genotype in cattle [10, 12, 13, 21]. One study diagnosed G. enterica more frequently than G. bovis[26] whereas studies in New Zealand found only infections with G. duodenalis and G. enterica, but not with G. bovis[2729].

The finding of sub-genotypes E2 and E3 within the species G. bovis (Assemblage E) is similar to former studies [11, 14, 21]. According to Xiao and Fayer [30] and Feng and Xiao [1] A1 and A2 are the most common sub-genotypes of G. duodenalis (Assemblage A), with humans being mostly infected with A2 and animals with A1. This agrees with former results [14, 22, 23] and with the results of this study, as A1 was the only sub-genotype of G. duodenalis diagnosed. However, others have found one or more of the sub-genotypes A1-A4 in cattle [1012, 21, 24]. Therefore it is possible that calves can be infected with a variety of sub-genotypes of G. duodenalis, all of which have also been identified in humans [21]. This suggests that there may be an interaction between the human and livestock transmission cycle [3]. Cattle have long been assumed to be of public health significance as a source of waterborne outbreaks of giardiasis in humans due to contamination of ground and surface water, although, there is no evidence incriminating infected cattle in any of the 132 documented waterborne outbreaks [2]. However, it has been shown, that animal handlers can be in danger of zoonotic transmission of G. duodenalis from infected cattle [14], and in reverse anthropozoonotic transmission of G. duodenalis from animal handlers to cattle is also possible [13]. Thus, transmission of the zoonotic species, which was detected in this study, could in principle be possible between animal handlers and cattle.

The role of Giardia as a cause of diarrhoea in calves is still unclear, as there are conflicting results from a number of studies, some demonstrating an association and others not. Furthermore, the presence of species-specific pathogenicity in calves poses further difficulties in the evaluation and has not been determined in another bovine study [11]. The role of the particular Giardia-species in mixed-infections in diarrhoeic calves could not be clarified either. However, the identification of some diarrhoeic samples, where G. bovis was the only pathogen detected, may suggest that this species does contribute to diarrhoea in calves. Whether these results are indicative or not remains unclear. Further studies will show whether differences in the clinical outcomes can occur due to the various sub-genotypes as has been established in human medicine [2].

Conclusions

The results of this study show that although the livestock specific species G. bovis has been diagnosed most frequently, the potential zoonotic species G. duodenalis is also present in calves in Southern Germany and thus might be a risk for animal handlers. Furthermore the results indicate that G. bovis might contribute to diarrhoea, as it was the only pathogen found in a proportion of the samples from diarrhoeic calves.

Declarations

Acknowledgements

We thank our colleagues in the lab, especially Elisabeth Kiess, Kathrin Simon and Tim Tiedemann for their contribution to the study.

Authors’ Affiliations

(1)
Comparative Tropical Medicine and Parasitology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München
(2)
School of Veterinary and Biomedical Sciences, Murdoch University

References

  1. Feng Y, Xiao L: Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011, 24: 110-140. 10.1128/CMR.00033-10.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Thompson RC, Monis P: Giardia-from genome to proteome. Advances in Parasitology. Volume 78. Edited by: Rollinson D, Hay SI. 2012, London: Elsevier, 57-95.View ArticleGoogle Scholar
  3. Monis PT, Caccio SM, Thompson RC: Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol. 2009, 25: 93-100. 10.1016/j.pt.2008.11.006.View ArticlePubMedGoogle Scholar
  4. Geurden T, Vercruysse J, Claerebout E: Field testing of a fenbendazole treatment combined with hygienic and management measures against a natural Giardia infection in calves. Vet Parasitol. 2006, 142: 367-371. 10.1016/j.vetpar.2006.07.019.View ArticlePubMedGoogle Scholar
  5. Geurden T, Vercruysse J, Claerebout E: Is Giardia a significant pathogen in production animals?. Exp Parasitol. 2010, 124: 98-106. 10.1016/j.exppara.2009.03.001.View ArticlePubMedGoogle Scholar
  6. Xiao L: Giardia infection in farm animals. Parasitol Today. 1994, 10: 436-438. 10.1016/0169-4758(94)90178-3.View ArticlePubMedGoogle Scholar
  7. O’Handley RM, Cockwill C, McAllister TA, Jelinski M, Morck DW, Olson ME: Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J Am Vet Med Assoc. 1999, 214: 391-396.PubMedGoogle Scholar
  8. Ralston BJ, McAllister TA, Olson ME: Prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams. Vet Parasitol. 2003, 114: 113-122. 10.1016/S0304-4017(03)00134-1.View ArticlePubMedGoogle Scholar
  9. Becher KA, Robertson ID, Fraser DM, Palmer DG, Thompson RC: Molecular epidemiology of Giardia and Cryptosporidium infections in dairy calves originating from three sources in Western Australia. Vet Parasitol. 2004, 123: 1-9. 10.1016/j.vetpar.2004.05.020.View ArticlePubMedGoogle Scholar
  10. Mendonca C, Almeida A, Castro A, de Lurdes DM, Soares S, da Costa JM, Canada N: Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal. Vet Parasitol. 2007, 147: 47-50. 10.1016/j.vetpar.2007.03.019.View ArticlePubMedGoogle Scholar
  11. Geurden T, Geldhof P, Levecke B, Martens C, Berkvens D, Casaert S, Vercruysse J, Claerebout E: Mixed Giardia duodenalis assemblage A and E infections in calves. Int J Parasitol. 2008, 38: 259-264. 10.1016/j.ijpara.2007.07.016.View ArticlePubMedGoogle Scholar
  12. Ng J, Yang R, McCarthy S, Gordon C, Hijjawi N, Ryan U: Molecular characterization of Cryptosporidium and Giardia in pre-weaned calves in Western Australia and New South Wales. Vet Parasitol. 2011, 176: 145-150. 10.1016/j.vetpar.2010.10.056.View ArticlePubMedGoogle Scholar
  13. Dixon B, Parrington L, Cook A, Pintar K, Pollari F, Kelton D, Farber J: The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada. Vet Parasitol. 2011, 175: 20-26. 10.1016/j.vetpar.2010.09.032.View ArticlePubMedGoogle Scholar
  14. Khan SM, Debnath C, Pramanik AK, Xiao L, Nozaki T, Ganguly S: Molecular evidence for zoonotic transmission of Giardia duodenalis among dairy farm workers in West Bengal, India. Vet Parasitol. 2011, 178: 342-345. 10.1016/j.vetpar.2011.01.029.View ArticlePubMedGoogle Scholar
  15. Geurden T, Vanderstichel R, Pohle H, Ehsan A, von Samson-Himmelstjerna G, Morgan ER, Camuset P, Capelli G, Vercruysse J, Claerebout E: A multicentre prevalence study in Europe on Giardia duodenalis in calves, with molecular identification and risk factor analysis. Vet Parasitol. 2012, 190: 383-390. 10.1016/j.vetpar.2012.06.039.View ArticlePubMedGoogle Scholar
  16. Heine J: Eine einfache Nachweismethode für Kryptosporidien im Kot. Zentralbl Veterinaermed Reihe B. 1982, 29: 324-327.View ArticleGoogle Scholar
  17. Thornton SA, West AH, DuPont HL, Pickering LK: Comparison of methods for identification of Giardia lamblia. Am J Clin Pathol. 1983, 80: 858-860.PubMedGoogle Scholar
  18. Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, Thompson RC: Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol. 1997, 83: 44-51. 10.2307/3284315.View ArticlePubMedGoogle Scholar
  19. Read CM, Monis PT, Thompson RC: Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect Genet Evol. 2004, 4: 125-130. 10.1016/j.meegid.2004.02.001.View ArticlePubMedGoogle Scholar
  20. Caccio SM, De Giacomo M, Pozio E: Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 2002, 32: 1023-1030. 10.1016/S0020-7519(02)00068-1.View ArticlePubMedGoogle Scholar
  21. Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Caccio SM: Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol. 2005, 35: 207-213. 10.1016/j.ijpara.2004.10.022.View ArticlePubMedGoogle Scholar
  22. Langkjaer RB, Vigre H, Enemark HL, Maddox-Hyttel C: Molecular and phylogenetic characterization of Cryptosporidium and Giardia from pigs and cattle in Denmark. Parasitology. 2007, 134: 339-350. 10.1017/S0031182006001533.View ArticlePubMedGoogle Scholar
  23. Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, Gregori F, Soares RM: Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of Sao Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol. 2007, 149: 258-264. 10.1016/j.vetpar.2007.08.019.View ArticlePubMedGoogle Scholar
  24. Feng Y, Ortega Y, Cama V, Terrel J, Xiao L: High intragenotypic diversity of Giardia duodenalis in dairy cattle on three farms. Parasitol Res. 2008, 103: 87-92.View ArticlePubMedGoogle Scholar
  25. Berrilli F, Di Cave D, De Liberato C, Franco A, Scaramozzino P, Orecchia P: Genotype characterisation of Giardia duodenalis isolates from domestic and farm animals by SSU-rRNA gene sequencing. Vet Parasitol. 2004, 122: 193-199. 10.1016/j.vetpar.2004.04.008.View ArticlePubMedGoogle Scholar
  26. Coklin T, Farber J, Parrington L, Dixon B: Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada. Vet Parasitol. 2007, 150: 297-305. 10.1016/j.vetpar.2007.09.014.View ArticlePubMedGoogle Scholar
  27. Winkworth CL, Learmonth JJ, Matthaei CD, Townsend CR: Molecular characterization of Giardia isolates from calves and humans in a region in which dairy farming has recently intensified. Appl Environ Microbiol. 2008, 74: 5100-5105. 10.1128/AEM.00232-08.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Learmonth JJ, Ionas G, Pita AB, Cowie RS: Identification and genetic characterisation of Giardia and Cryptosporidium strains in humans and dairy cattle in the Waikato Region of New Zealand. Water Sci Technol. 2003, 47: 21-26.PubMedGoogle Scholar
  29. Hunt CL, Ionas G, Brown TJ: Prevalence and strain differentiation of Giardia intestinalis in calves in the Manawatu and Waikato regions of North Island, New Zealand. Vet Parasitol. 2000, 91: 7-13. 10.1016/S0304-4017(00)00259-4.View ArticlePubMedGoogle Scholar
  30. Xiao L, Fayer R: Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol. 2008, 38: 1239-1255. 10.1016/j.ijpara.2008.03.006.View ArticlePubMedGoogle Scholar

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© Gillhuber et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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