Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal

https://doi.org/10.1016/j.vetpar.2007.03.019Get rights and content

Abstract

Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin–ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and β-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.

Introduction

Giardia spp. and Cryptosporidium spp. are the most common protozoan parasites that infect domestic animals and humans (Fayer, 2004, Thompson, 2004, Thompson and Monis, 2004, Appelbee et al., 2005).

Cryptosporidiosis causes significant neonatal morbidity in cattle, causing weight loss and delayed growth, which leads to large economic losses (McDonald, 2000). In immunocompetent humans the disease is usually self-limiting, however, in immunocompromised patients is frequently a chronic, severe, and sometimes fatal infection (Fayer, 2004). Cryptosporidiosis is a common secondary infection in patients with Acquired Immuno-Deficiency Syndrome (AIDS) that results in twice as many fatalities as any other AIDS associated infections (Colford et al., 1996). Cryptosporidium is both a waterborne and a foodborne pathogen that can survive for months outside its host. Two species of Cryptosporidium have been identified in cattle: C. parvum in the intestine and C. andersoni in the abomasum. C. parvum infects cattle and other mammals including humans (Thompson, 2003).

Giardia is an ubiquitous enteric parasite that affects domestic animals and humans (Thompson et al., 2000). Giardia duodenalis has been implicated as an etiological agent in dairy and beef calf diarrhea, worldwide (O’Handley et al., 1999, Huetink et al., 2001, Olson et al., 2004). Three genotypes of G. duodenalis cause infection in cattle: the zoonotic genotype, assemblages A and B and the livestock genotype, assemblage E (Olson et al., 2004, Trout et al., 2006).

Molecular characterization of Giardia and Cryptosporidium has given rise to a clearer epidemiological picture, with better information on the zoonotic potential and transmission mechanisms for humans; nevertheless the frequency of zoonotic transmission is still poorly understood (Thompson, 2004, Cacciò et al., 2005). Additional molecular epidemiological studies are needed to determine the frequency and the importance of the zoonotic transmission.

In the present study, were isolate Cryptosporidium and Giardia from cattle feces in order to perform molecular studies with the objective of contribute for clarifying the epidemiology of those infections.

Section snippets

Sampling

All animals used in this study were from commercial farms in the two main cattle raising regions of central and northern Portugal “Beira Litoral” and “Entre Douro e Minho”, comprising a population of approximately 490,000 dairy and beef cattle. Those regions are densely populated and have three main rivers: “Cávado”, “Ave” and “Vouga” running through. Fecal samples were collected from 66 dairy farms ranging from 5 to 310 animals and from 30 beef farms ranging from 2 to 45 animals. Of the 467

Results

Seventy-four out of 291 calves (25.4%) and 8 out 176 adults (4.5%) were positive for Cryptosporidium. Forty-one out 291 calves samples (14.1%) and 1 out 176 adult samples (0.57%) were positive for Giardia. Additionally, in the calves sampled, 12 out of 291 (4.1%) were positive for both Cryptosporidium and Giardia, however, no co-infections were detected in the adults.

From the 82 Cryptosporidium positive samples we obtained 63 isolates from calves and 7 isolates from adults. Additionally, from

Discussion

Human cryptosporidiosis is mainly caused by C. hominis and C. parvum (Kosek et al., 2001). The results in the present study showed that the C. parvum was more prevalent in cattle from Portugal, which is in agreement with previous reports from Portugal (Alves et al., 2003, Alves et al., 2006). The zoonotic implications of those finding should be take into account for better understand the epidemiology of cryptosporidiosis in the country.

Molecular characterization of the Cryptosporidium isolates

Acknowledgements

We gratefully acknowledge the revision of this manuscript by Dr. Stephen Kane and by Professor Bruce Eilts. Fundação Calouste Gulbenkian, project number 61018, financially supported this work.

Cited by (69)

  • Prevalence and molecular characterization of Giardia duodenalis isolates from dairy cattle in northeast China

    2015, Experimental Parasitology
    Citation Excerpt :

    As one of the two major assemblages of G. duodenalis causing human giardiosis, assemblage A was found in humans as well as in cattle. This type of occurrence is quite common in a few countries, such as 50% in Italy (Lalle et al., 2005), 42.9% in Canada (Uehlinger et al., 2006) and 14.3% in Portugal (Mendonça et al., 2007). In New Zealand, assemblage A was found to be the major G. duodenalis genotype (54.2–87.5%) (Hunt et al., 2000; Learmonth et al., 2003; Winkworth et al., 2008).

  • A Perspective on cryptosporidium and giardia, with an emphasis on bovines and recent epidemiological findings

    2015, Advances in Parasitology
    Citation Excerpt :

    However, zoonotic assemblage A is increasingly being detected in cattle, indicating that this assemblage might be more widespread in cattle than previously assumed (reviewed in Ryan and Cacciò, 2013). In contrast, assemblage B has been reported from cattle only on some occasions (Coklin et al., 2007; Lalle et al., 2005b; Mendonca et al., 2007; Ng et al., 2011; Winkworth et al., 2008). A long-term survey in the UK (Bodley-Tickell et al., 2002) found Cryptosporidium in surface water draining from a livestock farm and reported the presence of oocysts throughout the year; the highest levels coincided with the calving season.

View all citing articles on Scopus
View full text