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Parasitol Res (2009) 105:141–144 DOI 10.1007/s00436-009-1374-4

ORIGINAL PAPER

A longitudinal study of Enterocytozoon bieneusi in dairy cattle Mónica Santín & Ronald Fayer

Received: 16 December 2008 / Accepted: 6 February 2009 / Published online: 4 March 2009 # Springer-Verlag 2009

Abstract Feces from each of 30 Holstein cattle on a Maryland dairy farm were examined at weekly, bimonthly, and then monthly intervals from 1 week to 24 months of age for the presence of Enterocytozoon bieneusi. DNA was extracted from spores cleaned of fecal debris, and a twostep nested PCR protocol was used to amplify a fragment of the internal transcriber spacer region of the rRNA gene. All PCR-positive specimens were sequenced to determine the genotype of E. bieneusi. The overall prevalence was 24% (239/990) with a lower prevalence in pre-weaned calves (less than 8 weeks of age; 11.7%) and heifers (13– 24 months of age) than post-weaned calves (3–12 months of age; 44.4%). Over the course of 24 months, the cumulative prevalence of E. bieneusi was 100% since all 30 calves shed spores at some time during the study. One or more of three genotypes of E. bieneusi, J, I, and BEB4, were detected in all 30 animals. Genotype I was detected in all 30 cattle between 1 week and 22 months of age with some cattle remaining infected as long as 17 months. At 4 months of age, 28 cattle were infected with genotype I. Genotype BEB4 was detected briefly in seven cattle, most between 15 and 20 months of age. Genotype J was detected in eight cattle, all between 16 and 24 months of age. This longitudinal study strongly supports the findings of point prevalence, multiple farm studies in which genotypes J, I, and BEB 4 were found. These genotypes appear to be cattle M. Santín : R. Fayer (*) Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA e-mail: [email protected]

specific and have not been found in humans or other animals.

There are now over 80 genotypes of Enterocytozoon bieneusi reported based on the nucleotide sequence of the ITS region (Santín and Fayer 2009), the species of Microsporidia reported most frequently in humans with microsporidiosis (Reetz et al. 2002). They have been found in a wide variety of livestock, domesticated animals, and wild mammals and most appear host specific (Breitenmoser et al. 1999; Buckholt et al. 2002; Dengjel et al. 2001; Desplazes et al. 1996; Fayer et al. 2003; Lores et al. 2002; Mathis et al. 1999; Reetz et al. 2002; Rinder et al. 2000; Santín et al. 2004, 2005; Sulaiman et al. 2003). The first report of E. bieneusi in cattle was for a few calves in Germany (Dengjel et al. 2001; Rinder et al. 2000) and the most recent was for a few others in Korea (Lee 2007). A series of four larger studies was conducted on Holstein cattle on 14 dairy farms in seven states along the East Coast of the USA from Vermont to Florida. Of 413 pre-weaned dairy calves, 3.1% were found PCR positive for E. bieneusi (Fayer et al. 2003). Of 452 post-weaned, 3- to 8-month-old calves, 13% were found positive for E. bieneusi (Santín et al. 2004). Of 571 heifers, 12 to 24 months of age, 23% were positive for E. bieneusi (Santín et al. 2005). Of 541 milking cows, 4.4% were positive for E. bieneusi (Fayer et al. 2007). All of the foregoing reports presented data from point prevalence studies that captured a moment in time for each animal at each location. The present study is the first longitudinal study of E. bieneusi and the objective was to determine the prevalence of E. bieneusi at the molecular level in the same cattle from birth to 24 months of age on a single farm.

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Materials and methods Thirty purebred Holstein female calves born on a dairy farm in Maryland over a period of 7 months were individually monitored from 1 week to 2 years of age. Newborn calves were fed colostrum and received injections of Penicillin G Benzathine and Penicillin G Procaine, Bo– Se (selenium), vitamins A, C, and D, and iron dextran. They were housed in individual outdoor hutches. Clean straw bedding was provided daily and calves were fed 750 ml of CalfMaker® milk replacer (Southern States, Richmond, VA, USA) supplemented with a vitamin– mineral mixture twice daily until they reached 6 weeks of age. Thereafter, calves were moved to large communal pens with cement floors where alfalfa, timothy hay, orchard grass hay, and/or calf starter and fresh water were available ad libitum. Feces were collected directly from the rectum into plastic specimen cups at weekly intervals from 1 through 8 weeks of age, at bi-weekly intervals from 3 through 5 months of age (weeks 10, 12, 14, 16, 18, and 20), and at monthly intervals thereafter until they reached 24 months of age. Feces were held at 5°C within 1 h after collection and within 1–3 days, 15 g of feces from each calf was sieved and subjected to CsCl density gradient centrifugation to remove most debris and concentrate spores as described (Fayer et al. 2003). PCR amplification was performed with nested primers for E. bieneusi that amplified the ITS region and a portion of the flanking large and small subunit ribosomal RNA genes (Buckholt et al. 2002). All PCR protocols were identical to those used by Santín et al. (2005). Negative and positive controls were included in all PCR sets. The negative controls were also amplified from the first PCR in the second reaction to test for contamination. All PCRpositive samples were sequenced with the EBITS1/ EBITS2.4 primer pair. Products were purified with EXOSap enzyme (USB Corporation, Cleveland, OH, USA). Purified products were sequenced as described using BigDye chemistries and an AB 3100 sequence analyzer (Applied Biosystems, Foster City, CA, USA). Sequence chromatograms for each ∼390 bp strand were aligned and inspected with the aid of Lasergene software (DNASTAR Inc., Madison, WI, USA). These sequences were compared with those in Genbank by BLAST analysis.

Results Of the 30 calves, 100% were positive for E. bieneusi at some time during the study based on PCR (Table 1). None of the infected animals had diarrhea or appeared ill. Of 990 fecal specimens collected, 239 (24%) were PCR positive

Parasitol Res (2009) 105:141–144 Table 1 Enterocytozoon bieneusi-positive specimens identified by PCR for each of the calves examined at weekly (1–8 weeks), biweekly (10–20 weeks), and monthly (6–24 months) intervals from 1 week to 24 months of age Calf ID

Positive specimens (genotype)

1

5 6

4w(I), 5w(I), 14w(I), 16w(I), 6m(I), 9m(I), 13m(I), 17m(I), 19m(I) 6w(I), 12w(I), 14(I), 18w(I), 20(I), 7m(I), 8m(I),18m(I), 19(I), 24m(J) 1w(BEB4), 3–7w(BEB4), 12–20w(I), 6m(I), 8m(I), 11m(I), 14m(I), 17m(I), 18m(I), 20m(J), 24(J) 4–8w(I), 12–18w(I), 6m(I), 7m(I), 14m(I), 16m(I), 17m(I), 19m(I) 1–7w(I), 12w(I), 14w(I), 18w(I), 20w(I), 11m(I), 12m(I) 14w–18w(I), 17m(BEB4)

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

10w(I), 14–20w(I), 11m(I), 12(I) 14–20w(I), 12–14m(I) 8w(I), 14–20w(I), 7m(I), 10m(I), 11m(I), 13m(I), 20m(J) 16w(I), 18w(I), 6m(I), 7m(I), 14m(I) 16–20w(I), 7m(I), 13m(I) 5w(I), 12–18w(I), 7m(I), 10m(I), 12m(I), 13m(I) 12–16w(I), 11m(I), 12m(I), 22m(J) 12–16w(I), 6m(I), 7m(I), 9m(I), 10m(I), 22m(J) 4w(I), 14–18w(I), 6m(BEB4), 9m(BEB4), 11m(I), 12m(I) 10–16w(I), 20w(I), 6m(I), 7m(I), 9–11m(I), 14–16m(I) 10–16w(I), 10m(I), 11m(I) 10–16w(I), 11m(I), 16m(BEB4) 10–20w(I), 8m(I), 9m(I), 11m(I), 15m(BEB4) 10–14w(I), 18w(I), 8m(I), 9m(I), 11m(I), 24m(J) 5–7w(I), 18w(I), 20w(I), 7m(I), 11m(I) 18w(I), 20w(I), 8–11m(I), 22m(I) 12w(I), 16w(I), 9m(I), 10m(I), 20m(BEB4) 1w(I), 12–16w(I), 7m(I), 8m(I), 10m(I), 15m(BEB4) 10–20w(I), 16m(J), 22m(J)

26 27 28 29 30

12–16w(I), 20w(I), 7m(I), 10w(I), 12w(I), 16w(I), 7m(I), 8m(I) 10w(I), 12w(I), 16w(I), 17m(J), 18(J) 12w(I), 19–20w(I), 6–8m(I) 12–16w(I), 8m(I)

2 3 4

In parentheses is the E. bieneusi genotype identified in each specimen, genotype I, J, or BEB4

for E. bieneusi. Prevalence varied with the age of the calves. Post-weaned calves had the highest prevalence of infection, especially during 3 and 5 months of age (weeks 10–20), and pre-weaned calves had the lowest prevalence of infection, especially from 1 to 3 weeks of age (Table 1, Fig. 1). Three genotypes of E. bieneusi were detected based on the nucleotide sequence of the ITS region of the rRNA

Parasitol Res (2009) 105:141–144

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Fig. 1 Prevalence of E. bieneusi and E. bieneusi genotypes I, J, and BEB4 in calves from 1 week to 24 months of age

100

Pre-weaned calves

Post-weaned calves

Heifers

Prevalence (%)

80

60

40

20

24 mo

22 mo

20 mo

18 mo

16 mo

14 mo

12 mo

10 mo

8 mo

6 mo

18 wk

14 wk

10 wk

7 wk

5 wk

3 wk

1 wk

0

Age E.bieneusi

gene, genotype J, genotype I, and genotype BEB4. Based on a consensus report when multiple names appear for the same ITS sequence, the first recorded name for that sequence will be considered the primary name and genotype names published thereafter for the same sequence will become junior synonyms (Santín and Fayer 2009). Genotype I (AF135836) is now the primary name for BEB2 (AY331006) and CEbE (EF139199), and genotype J is now the primary name for BEB1 (AY331005), PtEb X (DQ885586), and CEbB (EF139196). The three E. bieneusi genotypes identified in the present study differed in prevalence, duration of infection, and general age of the calves at the time of infection. Genotype I, the most prevalent genotype, was present for a much longer duration than the other genotypes, and (with one exception) was found in younger calves than the other genotypes. Twenty four calves were infected with genotype I for 9 months or longer. Of those, two were infected for 18 months and two others for 19 months. Between 1 and 3 weeks of age, two calves were infected with genotype I. Between 4 and 8 weeks of age, eight calves were infected with genotype I. Between 3 and 5 months of age, all 30 calves were infected with genotype I. Between 6 and 11 months of age, seven to 13 calves were still infected with genotype I. From 12 to 19 months, the number of calves infected with genotype I was reduced from six to three and thereafter only one calf was found infective at 22 months of age. Genotype BEB4 was detected in seven calves for short durations. One calf was infective from 1 to 7 weeks of age, another from 6 to 9 months of age, and each of the remaining five were infected only at a single time period between 15 and 20 months of age. Genotype J was found in eight calves between 16 and 24 months of age. Three calves were found

J

I

BEB4

infected at 2 monthly intervals and the remaining five were infected only at a single time period.

Discussion Each of 30 calves born on the same farm at varying intervals over a period of 7 months was examined at 33 intervals from 1 week to 24 months of age resulting in the testing of 990 fecal specimens. This first longitudinal study of microsporidiosis in cattle has demonstrated an extremely high prevalence of infection with E. bieneusi. The cumulative prevalence of infection increased from 10 of 30 (33%) pre-weaned calves (1–8 weeks of age) to all 30 (100%) post-weaned calves (3 to 12 months of age) and then decreased to 24 of 30 (80%) heifer calves (13– 24 months of age) (Table 1). Three point prevalence studies of dairy calves on 14 farms in six states reported the prevalence of E. bieneusi infections in calves of similar age to those in the present study (Fayer et al. 2003; Santín et al. 2004, 2005). However, differences between the longitudinal study and the point prevalence studies were found with regard to the prevalence of infection at different ages. The prevalence of infection found in calves in the point prevalence studies increased from 3% of pre-weaned to 13% of post-weaned to 23% of 12- to 24-month-old calves. However, amongst farms in those studies, there was great variation, which renders attempts at direct comparison inaccurate. For example, the percent of infected preweaned calves on each farm ranged from 0 to 23.5%, while the percent of infected post-weaned calves ranged from 0 to 34.7%, and the percent of infected 12- to 24-month-old calves ranged from 4.7% to 37.8% (Fayer et

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al. 2003; Santín et al. 2004, 2005). Variation was also observed within farms. For example, a farm in Vermont had a higher percent of pre-weaned calves versus post-weaned infected (23.5 vs. 7.69%) whereas a nearby farm had a lower percent of pre-weaned calves versus post-weaned infected (0 vs. 11.1%) (Santín et al. 2004). As calves increased in age from pre-weaned, to post-weaned, to 12 to 24 months of age, the percent of farms with infected calves in those age groups increased from 43% to 79% to 100%. If calves were examined on a different day or season, the results of the point prevalence studies might differ. We do not have an explanation for the increase of E. bieneusi prevalence in post-weaned calves. However, it could be attributed to the longer exposure time of post-weaned calves to parasites in the environment perhaps facilitated by a change from individual housing provided for pre-weaned calves to group housing of post-weaned calves and heifers. Other possibilities are the change in the diet: milk versus pelleted or grain-based feed, or the physiological differences associated with single stomach digestion in pre-weaned calves versus rumen function in older animals. Genotypes J, I, and BEB4 were the only genotypes found in calves in the present study and appear to be cattle specific since they have not been found in humans or other animals. The presence of each of these genotypes during the study varied with the age of the calves, the number of calves infected, and the duration of infection. Genotype I was the dominant genotype, found in all age groups but primarily in postweaned calves, infecting all 30 calves, and present in each calf over the longest time. There are no comparable data reported for genotypes of E. bieneusi for cattle or other hosts. In other studies of cattle, other genotypes have also been identified: M (AF267143), N (AF267144), PtEb XI (DQ885587), BEB3 (AY331007), BEB6 (EU153584), BEB7 (EU153585), CEbA (EF139195), CEbD (EF139198), CEbF (EF139194), and 4948 FL-2 2004 (DQ154136), EbpA (AF076040), D (AF101200), Peru6 (AY371281), Type IV (AF242478) (Santín and Fayer 2009). The genotypes EbpA (AF076040), D (AF101200), Peru6 (AY371281), and Type IV (AF242478) have been reported not only in cattle but also in other hosts. As in point prevalence studies involving pre-weaned, post-weaned, and heifer calves (Fayer et al. 2003; Santín et al. 2004, 2005), there was no indication of diarrhea in any of the fecal specimens obtained from cattle, indicating that the E. bieneusi genotypes detected in these animals produced no detectable symptoms.

Parasitol Res (2009) 105:141–144 Acknowledgments The authors thank Kristin Cameron and Brooke Reich for technical services in support of this study.

References Breitenmoser AC, Mathis A, Bürgi E, Weber R, Desplazes P (1999) High prevalence of Enterocytozoon bieneusi in swine with four genotypes that differ from those identified in humans. Parasitology 118:447–453 Buckholt MA, Lee JH, Tzipori S (2002) Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. App Environ Microbiol 68:2595–2599 Dengjel B, Zahler M, Hermanns W, Heinritz K, Spillmann T, Thomschke A, Loscher T, Gothe R, Rinder H (2001) Zoonotic potential of Enterocytozoon bieneusi. J Clin Micro 39:4495– 4499 Desplazes P, Mathis A, Müller C, Weber R (1996) Molecular epidemiology of Encephalitozoon cuniculi and first detection of Enterozytozoon bieneusi in faecal samples of pigs. J Eukaryot Microbiol 43:93S Fayer R, Santín M, Trout JM (2003) First detection of microsporidia in dairy calves in North America. Parasitol Res 90:383–386 Fayer R, Santín M, Trout JM (2007) Enterocytozoon bieneusi in mature dairy cattle on farms in the eastern United Sates. Parasitol Res 102:15–20 Lee JH (2007) Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea. Parasitol Res 101:391–396 Lores B, del Aguila C, Arias C (2002) Enterocytozoon bieneusi (Microsporidia) in faecal samples from domestic animals from Galicia, Spain. Mem Inst Oswaldo Cruz 97:941–945 Mathis A, Breitenmoser AC, Desplazed P (1999) Detection of new Enterocytozoon genotypes in faecal samples of farm dogs and a cat. Parasite 6:189–193 Reetz J, Rinder H, Thomschke A, Manke H, Schwebs M, Bruderek A (2002) First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens). Int J Parasitol 32:785–787 Rinder H, Thomschke A, Dengjel B, Gothe R, Löscher T, Zahler M (2000) Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J Parasitol 86:185–188 Santín M, Fayer R (2009) Enterocytozoon bieneusi genotype nomenclature based on the ITS sequence—a consensus. J Eukaryot Microbiol 56 (1): in press Santín M, Trout JM, Fayer R (2004) Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves in the eastern United States. Parasitol Res 93:287–289 Santín M, Trout JM, Fayer R (2005) Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States. Parasitol Res 97:535–538 Sulaiman IM, Fayer R, La AA, Trout JM, Schafer FW, Xiao L (2003) Molecular characterization of microsporidia indicates that wild mammals harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi. Appl Environ Microbiol 69:4495–4501