Genetic uniqueness of Cryptosporidium parvum from dairy calves in Colombia.

Fecal specimens from 432 pre-weaned calves younger than 35 days were collected over a 2-year period (2010-2012) from 74 dairy cattle farms in the central area of Colombia. These samples were microscopically examined for the presence of Cryptosporidium oocysts, and positive specimens were selected for molecular examination. Microscopy revealed that 115 calves (26.6%) from 44 farms (59.5%) tested positive. Oocyst shedding was recorded in calves aged 3-day-old onwards, although the infection rate peaked at 8-14 days (40.7%). Infection rates were higher in diarrheic (52.2%) than in non-diarrheic calves (19.9%) (p < 0.0001, χ2), and infected calves had up to seven times more probability of having diarrhea than non-infected calves. Cryptosporidium species and subtypes were successfully identified in 73 samples from 32 farms. Restriction and sequence analyses of the SSU rRNA gene revealed C. parvum in all but two isolates identified as Cryptosporidium bovis. Sequence analyses of the 60-KDa glycoprotein (gp60) gene revealed eight subtypes within the IIa family. An unusual subtype (IIaA18G5R1) was the most prevalent and widely distributed (more than 66% specimens and 68% farms) while the subtype most frequently reported in cattle worldwide (IIaA15G2R1) was found in less than 13% of specimens and 16% farms. The remaining subtypes (IIaA16G2R1, IIaA17G4R1, IIaA20G5R1, IIaA19G6R1, IIaA20G6R1, and IIaA20G7R1) were restricted to 1-3 farms. This is the first large-sample size study of Cryptosporidium species and subtypes in Colombia and demonstrates the genetic uniqueness of this protozoan in cattle farms in this geographical area.

Cryptosporidium species and subtype analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain.

Faecal specimens from diarrhoeic pre-weaned lambs (n = 171) and goat kids (n = 118) were collected in 37 sheep and 23 goat flocks, respectively, from NW Spain and microscopically examined for the presence of Cryptosporidium oocysts. Positive specimens were selected for molecular characterization. Presence of Cryptosporidium oocysts were significantly higher in specimens from goat kids (62.7%) than from lambs (31.6%). PCR products of the SSU rRNA locus were obtained for 108 isolates, and three Cryptosporidium species were identified. Cryptosporidium parvum was the most common species identified from both lambs (74.4%) and goat kids (93.8%). The remaining PCR products from lambs (25.6%) and goat kids (7.7%) were identified as Cryptosporidium Ubiquitum and Cryptosporidium xiaoi, respectively. Five C. parvum subtypes were identified; IIaA13G1R1, IIaA14G2R1, IIaA15G2R1 and IIaA16G3R1 were found in both host species, and IIdA17G1 was only detected in goat kids. Subtype IIaA15G2R1 was the most common and widely distributed. The present study provides the first description of subtypes IIaA13G1R1 in both small ruminant species, IIaA14G2R1 in sheep and IIaA16G3R1 in goats. Our results also reveal that diarrhoeic pre-weaned lambs and goat kids must be considered important reservoirs of Cryptosporidium species with zoonotic potential, such as C. parvum and C. ubiquitum.

IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis.

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

Validation of fragment analysis by capillary electrophoresis to resolve mixed infections by Cryptosporidium parvum subpopulations.

The potential of capillary electrophoresis (CE)-based DNA fragment analysis to identify mixed infections by Cryptosporidium parvum subpopulations was validated using high-resolution slab-gel electrophoresis. A selection of genomic DNA samples from C. parvum isolates with CE electropherogram profiles indicative of two concurrent alleles at one or more of six mini and microsatellite loci (MSB, MS5, ML1, ML2, TP14, 5B12) were analysed. These loci were PCR-amplified and products separated on precast Spreadex EL600 slab gels. ML1 PCR products differing by as little as 3 bp in length were visible after Spreadex gel electrophoresis and fragments were clearly separated for all but the ML2 and 5B12 loci, which generated stutter bands. No stuttering was seen for the remaining markers, having three or more nucleotide motifs in the repeat region. For each sample, the two bands of interest were excised separately, DNA extracted and re-amplified by PCR. Sequencing of these PCR products revealed the expected sequences for both alleles at most samples, except for the longest ML2 and 5B12 alleles which generated indeterminate sequences. Two novel MS5 alleles were successfully sequenced after PCR re-amplification. These findings demonstrate the utility of high-resolution Spreadex gels for analysing the polymorphism of satellite markers of Cryptosporidium isolates and support the validity of CE as a reliable and sensitive tool for detecting mixed Cryptosporidium subpopulations in a single-host infection.

Subtype Distribution of Blastocystis spp. in Patients with Gastrointestinal Symptoms in Northern Spain.

Limited molecular data exist on the prevalence and subtype distribution of Blastocystis spp., the most prevalent parasite in human and animal feces worldwide. A total of 44 different subtypes (STs) of Blastocystis are currently recognized based on the sequence of the small subunit ribosomal RNA (SSU-rRNA) gene. This is a molecular study of Blastocystis spp. in hospitalized patients with gastrointestinal symptoms in northern Spain. We analyzed 173 Blastocystis-positive patients with gastrointestinal symptoms by using nested PCR for molecular detection, subtype identification, phylogenetic analyses, and genetic diversity assessment. ST2 (34.1%) and ST3 (34.7%) predominated, followed by ST1 (15.6%) and ST4 (15.6%). Mixed infections with different subtypes were observed in some patients. Sequence analysis revealed for the first time in European humans the allele 88 (a variant of ST1). In other cases, alleles commonly found in animal samples were detected (allele 9 in ST2, allele 34 in ST3, and allele 42 in ST4). Phylogenetic analysis showed high variability in ST1 and ST2, suggesting a polyphyletic origin, while both ST3 and ST4 exhibited higher genetic homogeneity, indicating a possible monophyletic origin and recent transmission to humans. These data confirm Blastocystis spp. subtype diversity and may help in understanding the evolutionary processes and potential zoonotic transmission of this parasite.

Molecular Diagnosis of Footrot and Contagious Ovine Digital Dermatitis in Small Ruminants in the Iberian Peninsula.

Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols.

Host association of Cryptosporidium parvum populations infecting domestic ruminants in Spain.

A stock of 148 Cryptosporidium parvum DNA extracts from lambs and goat kids selected from a previous study examining the occurrence of Cryptosporidium species and GP60 subtypes in diarrheic lambs and goat kids in northeastern Spain was further characterized by a multilocus fragment typing approach with six mini- and microsatellite loci. Various degrees of polymorphism were seen at all but the MS5 locus, although all markers exhibited two major alleles accounting for more than 75% of isolates. A total of 56 multilocus subtypes (MLTs) from lambs (48 MLTs) and goat kids (11 MLTs) were identified. Individual isolates with mixed MLTs were detected on more than 25% of the farms, but most MLTs (33) were distinctive for individual farms, revealing the endemicity of cryptosporidial infections on sheep and goat farms. Comparison with a previous study in calves in northern Spain using the same six-locus subtyping scheme showed the presence of host-associated alleles, differences in the identity of major alleles, and very little overlap in MLTs between C. parvum isolates from lambs and those from calves (1 MLT) or isolates from lambs and those from goat kids (3 MLTs). The Hunter-Gaston index of the multilocus technique was 0.976 (95% confidence interval [CI], 0.970 to 0.982), which supports its high discriminatory power for strain typing and epidemiological tracking. Population analyses revealed the presence of two host-associated subpopulations showing epidemic clonality among the C. parvum isolates infecting calves and lambs/goat kids, respectively, although evidence of genetic flow between the two subpopulations was also detected.

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes.

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.

Prevalence and Associated Factors of Blastocystis sp. Infection in Patients with Gastrointestinal Symptoms in Spain: A Case-Control Study.

Blastocystis sp. is known to be the most prevalent parasite in fecal samples of humans worldwide. In the present report, a case-control study (1:9.89 (≈10)) was performed, by analyzing data from 3682 patients who attended a public hospital in the northern area of Spain showing gastrointestinal symptoms. Diagnosis was performed in human fecal samples by means of optical microscopy. The prevalence of Blastocystis sp. in patients with gastrointestinal symptoms was 9.18% (338/3682). Most of the Blastocystis sp.-infected patients tested negative for protozoa and helminths, and were underweight and foreign-born (26.4%), mainly from Africa and Central/South America. Gastrointestinal symptoms, such as abdominal pain, anorexia, halitosis, plus relative eosinophilia, as well as co-infections with pathogenic bacteria were associated with Blastocystis sp. infection. Both type 2 diabetes and treatment with immunosuppressive medicines at the time of Blastocystis sp. detection were associated with a higher proportion of infected patients. This is the first case-control study of Blastocystis sp. in humans in northern Spain and may contribute to surveillance and intervention strategies by public health authorities.

Low-cost ceramic membrane bioreactor: Effect of backwashing, relaxation and aeration on fouling. Protozoa and bacteria removal.

Membrane biological reactors (MBR) constitute an alternative to conventional wastewater treatments for improved recovery, reuse, and recycling of water. MBRs have a smaller footprint, provide better biotreatment and achieve a high-quality effluent. This work analyses the use of MBRs innovative low-cost ceramic membranes for wastewater treatment. We propose low-cost ceramic membranes as an alternative to the more expensive commercial ceramic membranes. Low-cost membranes were made of clay, calcium carbonate, potato starch, almond shell and chamotte. We synthesized two different selective layers, from clay and/or TiO2. We characterized the membranes (pore diameter and water permeance) and their performance in a laboratory scale MBR. To mitigate membrane fouling and preserve the continued operation along time, the effect of different operating cycles was measured, considering two physical cleaning strategies: relaxation and backwashing. Cycles of 9 min of operation, 30 s of relaxation and 1 min of backwashing provided the lowest fouling rate. We investigated the effect of air scouring on fouling by operating with different air flow rates. Once experimental conditions were optimized, the overall performance of the different ceramic membranes was tested. The membrane with a TiO2 thin layer provided the best resistance to fouling, as well as a good retention capacity of E. coli, Cryptosporidium oocysts and Giardia cysts.

Occurrence of Rotavirus A Genotypes and Other Enteric Pathogens in Diarrheic Suckling Piglets from Spanish Swine Farms.

Species A rotavirus (RVA) is a major viral pathogen causing diarrhea in suckling piglets. Studies on its genetic heterogeneity have implications for vaccine efficacy in the field. In this study, fecal samples (n = 866) from diarrheic piglets younger than 28 days were analyzed over a two-year period (2018-2019). Samples were submitted from 426 farms located in 36 provinces throughout Spain and were tested using real-time PCR (qPCR) and reverse transcription real-time PCR (RT-qPCR) for five enteric pathogens. The individual prevalence was 89.4%, 64.4%, 44.9%, 33.7% and 4.4% for Clostridiumperfringens, Clostridioides (formerly Clostridium) difficile, species A rotavirus, species C rotavirus and porcine epidemic diarrhea virus, respectively. Most specimens (96.9%) were positive for at least one of the target pathogens, and more than 80% of samples harbored mixed infections. Nucleotide sequencing of 70 specimens positive for RVA revealed the presence of the VP7 genotypes G4, G9, G3, G5, G11 and the VP4 genotypes P7, P23, P6 and P13, with the combinations G4P7 and G9P23 being the most prevalent, and especially in the areas with the highest pig population. The study shows the extensive genetic diversity of RVA strains as well as discrepancies with the genotypes contained in the vaccine available in Spain, and multiple amino acid differences in antigenic epitopes of different G- and P- genotypes with the vaccine strains. Further investigations are needed to determine the efficacy of the vaccine to confer clinical protection against heterologous strains.

Coxiella burnetii and Co-Infections with Other Major Pathogens Causing Abortion in Small Ruminant Flocks in the Iberian Peninsula.

Coxiella burnetii is an intracellular bacterium causing human Q fever and reproductive disorders in domestic ruminants. We analyzed the occurrence of C. burnetii and co-infections with six other major pathogens causing abortion in sheep (1242 cases) and goat (371 cases) flocks from Spain and Portugal. After real-time PCR detection, co-infections were established by principal component and cluster analysis that grouped cases based on the joint presence/absence of several microorganisms. C. burnetii and Chlamydia abortus were the most common abortifacient agents with approximately 75% of cases from both hosts testing positive, followed by Toxoplasma gondii, Campylobacter sp., Salmonella enterica, border disease virus and Neospora caninum. C. burnetii was significantly more common than C. abortus in goat abortions (p < 0.001). Co-infections with at least two pathogens were found in more than 66% cases of ovine abortions and 36% cases of caprine abortions testing positive for C. burnetii, mostly including mixed infections with only C. abortus. These findings indicate that both pathogens are the most significant ones to be readily prevented by vaccination in this geographical area. Biosecurity and biocontainment measures are also steadfastly recommended to prevent both the economic losses and public health risks associated with most of these abortifacient agents.

Multilocus fragment typing and genetic structure of Cryptosporidium parvum Isolates from diarrheic preweaned calves in Spain.

A collection of 140 Cryptosporidium parvum isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the C. parvum isolates from cattle farms in this geographical area.

Genotype and subtype analysis of Cryptosporidium isolates from calves and lambs in Galicia (NW Spain).

Faecal specimens from diarrhoeic pre-weaned calves (n=61) and lambs (n=127) collected over a 1-year period (2008-2009) at 27 cattle and 28 sheep farms in Galicia (NW Spain) were examined for the presence of Cryptosporidium oocysts and positive specimens were selected for molecular examination. Overall, 30 calves (49.2%) and 39 lambs (30.7%) tested positive for Cryptosporidium by microscopy. PCR products of the SSU rRNA locus were obtained for 27 Cryptosporidium positive calf isolates and 23 lamb isolates. Restriction analyses generated profiles of C. parvum in all isolates except for 9 lamb specimens from 5 farms that yielded banding patterns and sequences indicative of the Cryptosporidium cervine genotype. Sequence analyses of the glycoprotein (GP60) gene revealed that all but 1 C. parvum isolate from calves belonged to the subtype IIaA15G2R1 and 1 isolate was identified as the novel subtype IIaA13G1R1. Two different subtypes were identified in sheep flocks including IIaA16G3R1, which was seen in 7 lamb isolates from a single farm and subtype IIaA15G2R1, identified in 3 isolates from 2 farms. These findings suggest a limited genetic diversity within C. parvum in ruminant livestock from this geographical area, although both calves and lambs should be considered as a reservoir for zoonotic subtypes.

Occurrence and genetic diversity of rotavirus A in faeces of diarrheic calves submitted to a veterinary laboratory in Spain.

A total of 237 faecal specimens from diarrheic calves younger than two months were collected and submitted for diagnosis of enteropathogens over a two-year period (2017-2018) to a veterinary laboratory. Samples originated from 193 dairy and beef farms in 29 provinces distributed throughout Spain, and were tested for the occurrence of three target enteric pathogens by reverse transcription real-time PCR (RT-qPCR): bovine rotavirus A (RVA), Cryptosporidium parvum and bovine coronavirus (BCoV). RT-PCR and nucleotide sequencing analysis were used to determine the G (VP7 gene) and P (VP4 gene) genotypes of 26 specimens positive for RVA. A total of 188 specimens (79.3 %) were positive for at least one of the three target enteric pathogens, and 101 samples (42.6 %) harbored mixed infections. The individual prevalence was 57.8 %, 50.6 % and 23.6 % for C. parvum, RVA and BCoV, respectively. Molecular analysis of selected RVA strains revealed the presence of the G6, G10, G3, P[5] and P[11] genotypes, with the combinations G6P[5] and G6P[11] being the most prevalent. Alignments of nucleotide sequences of the VP7 and VP4 markers showed a high frequency of single nucleotide polymorphisms (SNPs), with up to 294 SNPs found in 869bp of sequence at the G6 genotype (0.338 SNPs/nt), which reveals the extensive genetic diversity of RVA strains. Phylogenetic analysis of the VP7 gene of the G6 strains revealed four distinct lineages, with most strains clustering in the G6-IV lineage. The discrepancies between the RVA genotypes circulating in the sampled cattle farms and the genotypes contained in commercial vaccines currently available in Spain are discussed. We believe that this is the first study on the molecular characterization of rotavirus infecting cattle in Spain.

Occurrence and molecular characterization of Giardia duodenalis in child population from Colombia.

Giardia duodenalis is one of the most prevalent human intestinal parasite, with children living in developing countries being particularly at risk of infection. The occurrence and molecular diversity of G. duodenalis was investigated in stools specimens from 307 individuals aged one to nineteen years in Colombia. Samples were collected in three educational establishments (n: 163) and two hospital laboratories (n: 144) from urban and rural areas. Feces were concentrated using a biphasic sedimentation method and wet mounts of the sediment were examined by light microscopy. G. duodenalis assemblages and sub-assemblages were determined on positive samples by PCR of the triose phosphate isomerase (tpi), ß-giardin (bg) and small-subunit (ssu) rRNA genes. G. duodenalis infection was detected by microscopy in 23 individuals (7.5%). The protozoan was more prevalent among specimens collected in educational establishments (11.6%) than in those obtained from hospital laboratories (2.8%). Infection was most common in individuals from urban areas and children aged 1-5 years. No significant association between diarrhea and infection could be demonstrated. Twenty Giardia-positive samples were successfully allocated to assemblage B (n: 11), sub-assemblage AII (n: 7), and assemblage A (n: 2). Results indicate the potential for transmission of G. duodenalis infection in children attending educational establishments and individuals from urban areas, where transmission seems to be primarily anthroponotic.

Multilocus fragment analysis of Cryptosporidium parvum from pre-weaned calves in Colombia.

The intra-species genetic diversity of Cryptosporidium parvum in dairy cattle farms in the central area of Colombia was investigated using a multilocus fragment typing approach with nine variable-number tandem-repeat (VNTR) loci and the gp60 gene. Genomic DNA of 70 C. parvum isolates from pre-weaned calves in 32 farms was analysed. Most markers showed two (ML1, MSB, CP47, and MSC6-7) or three alleles (5B12, Cgd2_3850, and Cgd6_5400), although they exhibited a major allele accounting for more than 69% of specimens, which explains their low discriminatory index. The TP14 microsatellite was monomorphic while a total of six alleles were found at the ML2 microsatellite. The two novel allelic variants (219bp, 245bp) exhibited by more than 36% of specimens at the latter locus were a remarkable finding. The 10-markers typing tool provided a Hunter-Gaston discriminatory value of 0.940 (95% CI, 0.918 - 0.961) and differentiated 22 multilocus subtypes (MLTs). Nevertheless, the combination of the three most informative markers (ML2, gp60, and Cgd2_3850) differentiated 68% of MLTs and hardly impaired the discriminatory index. The fact that many MLTs (13/22) were distinctive for individual farms provides evidence for the endemic nature of the infection and the major role played by transmission within farms. The eBURST algorithm suggested a low degree of genetic divergence. All but three MLTs were clustered in a clonal complex with a star-like topology typical of clonal expansion, however linkage analysis did not find evidence of linkage disequilibrium. Bayesian analysis also identified a genetic structure with K = 3 being the best estimation of ancestral clusters, although a large proportion of isolates (35%) could not be allocated to a single population, which indicates their mixed origin. The results confirm the genetic distinctiveness of C. parvum in cattle farms in this geographical area. This is the first multilocus analysis on the intra-specific variability of Cryptosporidium from calves in South America.

Cryptosporidium genotypes and subtypes in lambs and goat kids in Spain.

To provide information on the transmission dynamics of cryptosporidial infections in domestic small ruminants and the potential role of sheep and goats as a source for human cryptosporidiosis, Cryptosporidium-positive isolates from 137 diarrheic lambs and 17 goat kids younger than 21 days of age were examined by using genotyping and subtyping techniques. Fecal specimens were collected between 2004 and 2006 from 71 sheep and 7 goat farms distributed throughout Aragón (northeastern Spain). Cryptosporidium parvum was the only species identified by restriction analyses of PCR products from small-subunit rRNA genes from all 154 microscopy-positive isolates and the sequencing of a subset of 50 isolates. Sequence analyses of the glycoprotein (GP60) gene revealed extensive genetic diversity within the C. parvum strains in a limited geographical area, in which the isolates from lambs exhibited 11 subtypes in two subtype families (IId and IIa) and those from goat kids displayed four subtypes within the family IId. Most isolates (98%) belonged to the subtype family IId, whereas only three isolates belonged to the most widely distributed family, IIa. Three of the four most prevalent subtypes (IIdA17G1a, IIdA19G1, and IIdA18G1) were previously identified in humans, and five subtypes (IIdA14G1, IIdA15G1, IIdA24G1, IIdA25G1, and IIdA26G1) were novel subtypes. All IId subtypes were identical to each other in the nonrepeat region, except for subtypes IIdA17G1b and IIdA22G1, which differed by a single nucleotide polymorphism downstream of the trinucleotide repeats. These findings suggest that lambs and goat kids are an important reservoir of the zoonotic C. parvum subtype family IId for humans.

Cryptosporidium spp. CP15 and CSL protein-derived synthetic peptides' immunogenicity and in vitro seroneutralisation capability.

Cryptosporidium spp. is a zoonotic intracellular protozoan and a significant cause of diarrhoea in humans and animals worldwide. This parasite can cause high morbidity in immunocompromised people and children in developing countries, livestock being the main reservoir. This study was aimed at performing preliminary tests on Swiss albino weaned mice (ICR) to evaluate the humoral immune response induced against peptides derived from Cryptosporidium parvum CP15 (15 kDa sporozoite surface antigen) and CSL (circumsporozoite-like antigen) proteins. Peptides were identified and characterised using bioinformatics tools and were chemically synthesised. The antibody response was determined and the neutralising effect of antibodies was measured in cell culture. Despite all peptides studied here were capable of stimulating antibody production, neutralising antibodies were detected for just two of the CP15-derived ones. Additional studies aimed at evaluating further the potential of such peptides as vaccine candidates are thus recommended.

Molecular characterization of Cryptosporidium isolates from pigs in Zaragoza (northeastern Spain).

A total of 142 stool specimens from pigs on 24 farms from the province of Zaragoza (northeastern Spain) were screened for Cryptosporidium spp. Samples were first analysed by routine techniques (formalin-ethyl acetate sedimentation method and modified Ziehl-Neelsen stain) selecting those microscopically positive for genetic characterization. Cryptosporidium species and genotypes were determined by a nested PCR-RFLP technique at the 18S ribosomal DNA locus and sequencing of the PCR-positive secondary products. Cryptosporidium oocysts were microscopically identified in the faeces of 32 pigs (22.5%) from 15 farms (62.5%). Infected animals included 23 weaned piglets (30.7%), 5 fattening pigs (11.9%) and 4 sows (16%). Diarrhoea was not detected in any of the infected pigs. The molecular characterization was successfully performed in 26 samples from 14 farms. Cryptosporidium suis was found in 10 specimens from 7 farms (nine weaned piglets and one sow) and the Cryptosporidium pig genotype II in 16 samples from 10 farms (13 weaned piglets and 3 fattening pigs). Both C. suis and the pig genotype II were concurrently detected on three farms.