In order to determine the prevalence and risk factors for shedding of Cryptosporidium spp. in dairy calves, a cross-sectional study was carried out in the northeastern region of Buenos Aires Province, Argentina. Fecal samples from a total of 552 calves from 27 dairy herds were collected, along with a questionnaire about management factors. Cryptosporidium spp. oocysts were detected by light microscopy using Kinyoun staining. Putative risk factors were tested for association using generalized linear mixed models (GLMMs). Oocyst shedding calves were found in 67% (CI95% = 49–84) of herds (corresponding to a true herd prevalence of 98%) and 16% (CI95% = 13–19) of calves (corresponding to a true calve prevalence of 8%). Within-herd prevalence ranged from 0 to 60%, with a median of 8%. Cryptosporidium spp. excretion was not associated with the type of liquid diet, gender, time the calf stayed with the dam after birth, use of antibiotics, blood presence in feces, and calving season. However, important highly significant risk factors of oocyst shedding of calves was an age of less or equal than 20 days (OR = 7.4; 95% CI95% = 3–16; P < 0.0001) and occurrence of diarrhea (OR = 5.5; 95% CI95% = 2–11; P < 0.0001). The observed association with young age strongly suggests an early exposure of neonatal calves to Cryptosporidium spp. oocysts in maternity pens and/or an age-related susceptibility. Association with diarrhea suggests that Cryptosporidium spp. is an important enteropathogen primarily responsible for the cause of the observed diarrheal syndrome. Results demonstrate that Cryptosporidium spp. infection is widespread in the study region. Monitoring and control of this parasitic protozoan infection in dairy herds is recommended.

Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/ 1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ 2 = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocystpositive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.

No abstract

The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.Tetrahymena thermophila is a fresh-water protozoan that has been used successfully as a model system in cell biology (8). The advanced molecular and genetic tools developed for Tetrahymena have facilitated fundamental discoveries, such as the first descriptions of ribozymes, telomeres, and telomerases, thereby maintaining this organism at the forefront of fundamental research (2,11,30).Conner et al. (5, 6) described the peculiar sterol metabolism in Tetrahymena that leads to the accumulation of provitamin D analogs due to the C-5(6), C-7(8), and C-22(23) sterol-desaturating activities present in the organism (Fig. 1). The transformation of cholesterol to the C-7 unsaturated derivative (provitamin D 3 [cholest-5,7-dien-3␤-ol]) particularly has attracted attention because of pharmaceutical and food-related applications (28, 29) to decrease the cholesterol content in foodstuffs and the coupled production of provitamin D 3 in a single step (27). Despite the potential societal impact, progress on the isolation and purification of desaturases has been modest, mainly due to the loss of enzyme activity upon the dissociation of microsomal complexes (13).The preliminary characterization of sterol-desaturating activities in T. thermophila indicated that the corresponding enzymes are located in the microsomal fraction and require cytochrome (Cyt) b 5 , Cyt b 5 reductase, oxygen, and a reduced cofactor (NADH) (17). These biochemical requirements are characteristic of sterol C-5(6) desaturases and C-4 methyl oxidases (14). By using amino acid sequences of known C-5 desaturases as queries, eight putative desaturas...

The gene TTHERM_00438800 (DES24) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C 29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C 29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known.Sterols are lipophilic membrane components essential for the structural integrity of most eukaryotic cells. Together with phospholipids, they regulate the fluidity of the lipid bilayers and permeability barrier properties (4); they also serve as precursors of bile salts and a number of different steroid hormones in mammals, brassinosteroids in plants and fungi (2), and ecdysteroids in arthropods (14). Sterols are also actively involved in the modulation of cell signaling, in the transport and distribution of lipophilic molecules, and in the formation of lipid rafts (22).For most organisms in which sterols are de novo synthesized, such as vertebrates (cholesterol), plants (stigmasterol, ␤-sitosterol, and campesterol), and fungi (ergosterol), the enzymes involved have been well identified and characterized. Most of them can be grouped into four families of proteins: (i) the cytochrome b 5 (Cytb 5 )-dependent fatty acid hydroxylase superfamily, composed of C-5 sterol desaturases (erg3), C-4 sterol methyl oxidases (erg25), and cholesterol 25-hydroxylases; (ii) the S-adenosyl-L-methionine sterol methyltransferase (SMT) family, composed of the SMT1 and SMT2 types, which are typical in plants, and C-24 sterol methyltransferase, which has been described for fungi (erg6); (iii) the highly hydrophobic reductases, which include C-7, C-14, and C-24 sterol reductases; and (iv) the cytochrome P450 family, with C-22 sterol desaturases (erg5) and C-14 sterol demethylases (erg11) as its main representatives. Other eukaryotic org...