Detection and subtyping of influenza A virus in porcine clinical samples from Spain in 2020.

A total of 1019 samples collected on 726 Spanish swine farms suffering from outbreaks of respiratory disease were screened for influenza A viruses (IAVs) using a RT-qPCR method. A subset of positive samples was further analyzed using a subtype-specific RT-qPCR method (n: 142) and Sanger sequencing (n: 64). A total of 19.4% samples from 23% farms tested positive, with infection being most common in suckling (53.6%) and weaning pigs (30.2%). Viruses belonging to four HA subtypes (H1av, H1hu, H1pdm, H3) were detected, with subtypes H1avN2, H1huN2 and H1avN1 accounting for over half of the specimens. An optimized protocol with newly designed primers allowed the detection of H3 viruses in a significant number of samples (21%). A comparison of antigenic positions revealed that circulating strains exhibited mutations with vaccine strains in a significant percentage of amino acid residues, both in the NA protein (27.8-43.3%) and particularly in the HA protein (51-75.3%).

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.

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.

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.

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 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.

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.

Occurrence and genetic diversity of Cryptosporidium and Giardia in urban wastewater treatment plants in north-eastern Spain.

This study was designed to investigate the presence and removal efficiency of Cryptosporidium and Giardia in wastewater treatment plants at the 20 most populated towns in Aragón (north-eastern Spain). Samples of influent and effluent wastewater and dewatered sewage sludge were collected seasonally from 23 plants and processed according to USEPA Method 1623. All samples from raw and treated wastewater tested positive for Giardia, at an average concentration of 3247±2039cysts/l and 50±28cysts/l, respectively. Cryptosporidium was identified in most samples from both raw (85/92) and treated (78/92) wastewaters in a concentration significantly lower than Giardia, at both influent (96±105oocysts/l) and effluent samples (31±70oocysts/l) (P<0.001). The (oo)cyst counts peaked in summer in most plants. The removal efficiency was higher for Giardia (1.06-log to 2.34-log) than Cryptosporidium (0.35-log to 1.8-log). Overall, high removal efficiency values were found for Giardia after secondary treatment based on activated sludge, while tertiary treatment (microfiltration, chlorination and/or ultraviolet irradiation) was needed to achieve the greatest removal or inactivation of Cryptosporidium. Most samples of treated sludge were positive for Giardia (92/92) and Cryptosporidium (45/92), at an average concentration of 20-593cysts/g and 2-44oocyst/g, respectively. The molecular characterization of Cryptosporidium oocysts and Giardia cysts were attempted at the SSU rRNA/GP60 and bg/tpi loci, respectively. G. duodenalis sub-assemblage AII was identified in all plants, with a large proportion of samples (15/47) harboring mixed assemblages (AII+B). Nine Cryptosporidium species and six subtypes were identified, with C. parvum IIaA15G2R1 being the most prevalent. The presence of significant numbers of (oo)cysts in samples of final effluents and treated sludge reveals the limited efficacy of conventional treatments in removing (oo)cysts and highlights the potential environmental impact and public health risks associated with disposal and reclamation of wastewater.

Occurrence of Cryptosporidium and Giardia in raw and finished drinking water in north-eastern Spain.

This paper collects the first large-sample-size study on the presence of Cryptosporidium oocysts and Giardia cysts in drinking water plants at the 20 most populated towns in Aragón (north-eastern Spain). Samples of influent raw water and effluent finished water were collected from each plant during different seasons and processed according to USEPA Method 1623. Cryptosporidium oocysts and Giardia cysts were detected in samples collected from 55% and 70% plants, respectively, with nine plants being positive for both protozoa and only four plants being negative over the study period. Both parasites were identified in the raw water throughout the year, with a lower frequency in autumn and a peak in winter, at a mean concentration of 67±38 oocysts per 100l and 125±241 cysts per 100l. The turbidity of raw water was not related to the presence or concentration of (oo)cysts, and the (oo)cyst removal efficiency was not related to the type of water treatment. One or both pathogens were identified in the finished water in 7 out of 11 plants with a conventional treatment process (coagulation, flocculation, sedimentation, filtration, and disinfection processes) compared to 4 out of 9 plants that did not apply one of the pre-chlorination treatment steps. Protozoa were detected in the finished water of positive plants at a mean concentration of 88±55 oocysts per 100l and 37±41 cysts per 100l, and most of them excluded propidium iodide so were considered potentially viable. The ubiquity of these parasites in the drinking water sources and the inefficiency of conventional water treatment in reducing/inactivating them may present a serious public health issue in this geographical area.

Induction of protective immunity against experimental Eimeria tenella infection using serum exosomes.

Avian coccidiosis is caused by Eimeria, a unicellular, apicomplexan protist which primarily infects intestinal epithelia resulting in nutrient malabsorption and reduced growth of commercial poultry. Vaccination of chickens with exosomes isolated from antigen presenting cells containing parasite antigens (Ags) represents a promising alternative strategy to control avian coccidiosis, but is restricted in its commercial application due to limitations on production scale-up for mass immunization programs. Here, we report the biochemical and physiologic characteristics of exosomes derived from serum of Eimeria tenella-infected chickens and their feasibility for inducing protective immunity to experimental coccidiosis. Exosomes isolated from the serum of E. tenella-infected chickens contained a subset of protein Ags found in the intact parasite. Serum-derived exosomes containing these E. tenella Ags localized to the intestine and spleen following intramuscular injection into naïve chickens. In vitro ELISPOT assays revealed increased numbers of IL-2-, IL-4-, IL-6-, and IFN-γ-secreting cells in the intestine and spleen of exosome-administered chickens, compared with vehicle controls. Pre-immunization of chickens with serum exosomes from E. tenella-infected chickens increased both body weight gain and feed conversion efficiency, and reduced both fecal parasite shedding and gut lesion scores following parasite infection, compared with vehicle controls. Finally, immunization with CD80(+) serum exosomes stimulated greater numbers of cytokine-producing cells, and higher levels of protective immunity to E. tenella infection, compared with CD80(-) exosomes. These results suggest the possibility of producing an effective, parasite-free vaccine against avian coccidiosis under field conditions using serum-derived CD80(+) exosomes containing parasite Ags.

Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain.

A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7-9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979-0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population.

Intra-Species Diversity and Panmictic Structure of Cryptosporidium parvum Populations in Cattle Farms in Northern Spain.

The intra-herd and intra-host genetic variability of 123 Cryptosporidium parvum isolates was investigated using a multilocus fragment typing approach with eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene. Isolates were collected from intensively farmed diarrheic pre-weaned calves originating from 31 dairy farms in three adjoining regions in northern Spain (País Vasco, Cantabria and Asturias). The multilocus tool demonstrated an acceptable typeability, with 104/123 samples amplifying at all twelve loci. The ML2, TP14, GP60 and the previously un-described minisatellite at locus cgd2_3850 were the most discriminatory markers, while others may be dismissed as monomorphic (MSB) or less informative (CP47, ML1 and the novel minisatellites at loci Cgd1_3670 and Cgd6_3940). The 12-satellite typing tool provided a Hunter-Gaston index (HGDI) of 0.987 (95% CI, 0.982-0.992), and differentiated a total of 70 multilocus subtypes (MLTs). The inclusion of only the four most discriminatory markers dramatically reduced the number of MLTs (n: 44) but hardly reduced the HGDI value. A total of 54 MLTs were distinctive for individual farms, indicating that cryptosporidiosis is an endemic condition on most cattle farms. However, a high rate of mixed infections was detected, suggesting frequent meiotic recombination. Namely, multiple MLTs were seen in most farms where several specimens were analyzed (90.5%), with up to 9 MLTs being found on one farm, and individual specimens with mixed populations being reported on 11/29 farms. Bayesian Structure analysis showed that over 35% of isolates had mixed ancestry and analysis of evolutionary descent using the eBURST algorithm detected a high rate (21.4%) of MLTs appearing as singletons, indicating a high degree of genetic divergence. Linkage analysis found evidence of linkage equilibrium and an overall panmictic structure within the C. parvum population in this discrete 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.

Multilocus typing and population structure of Cryptosporidium from children in Zaragoza, Spain.

A multilocus typing approach with eight variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to analyze the inter- and intra-species variation of 44 Cryptosporidium isolates from pediatric patients in Zaragoza city (NE, Spain). Restriction and sequence analyses of the SSU rRNA gene revealed that Cryptosporidium transmission is mostly anthroponotic in this area, with the predominance of Cryptosporidium hominis (n: 41) over Cryptosporidium parvum (n: 3). GP60 subtyping showed limited genetic diversity and four subtypes were identified, including IbA10G2 (n: 35), IaA24R3 (n: 6), IIaA15G1R1 (n: 1) and IIaA15G2R1 (n: 2). Five out of eight VNTR loci showed a discriminatory power higher than the GP60 gene, although each locus had a predominant allele exhibited by more than 50% of isolates. All but four alleles were associated to either C. hominis or C. parvum and linked alleles at different loci were found. Multilocus typing substantially increased the discriminatory power (Hunter-Gaston index: 0.807, 95% CI, 0.683-0.926) and revealed that genetic diversity is much higher than that reported by GP60 sequencing, since 17 multilocus subtypes (MLTs) were identified. Nearly half of the specimens were allocated to a single major MLT. However, no more than three specimens were allocated to each of the remaining MLTs. Both phylogenetic and population analyses revealed a population clustering of C. hominis according to the GP60 subtype, which indicates the robustness of this marker to differentiate genetic subpopulations. Subpopulations had an overall clonal genetic structure, although traces of genetic flow between them were also observed.

Optimization of a fragment size analysis tool for identification of Cryptosporidium species and Gp60 alleles infecting domestic ruminants.

A capillary electrophoresis (CE)-based DNA fragment analysis tool was optimized to identify in a single capillary the most common Cryptosporidium species and Cryptosporidium parvum GP60 alleles infecting domestic ruminants. For this purpose, a panel of genomic DNA samples including six Cryptosporidium species (C. parvum, C. bovis, C. ryanae, C. andersoni, C. ubiquitum, and C. hominis) and 18 C. parvum GP60 subtypes belonging to the subtype families IIa and IId was used. All these samples had been characterized previously by sequencing of SSU rRNA and GP60 genes. Isolates were re-amplified by PCR at these loci using sets of newly designed primers and subjected to CE. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis. The optimized CE-based approach provided fragments of different size for most Cryptosporidium species, but did not differentiate C. bovis and C. ryanae. Many of the GP60 subtypes (11/18) were also readily differentiated by CE, although overlapping in fragment sizes between IIa and IId subtypes was noticed. The CE-based tool was subsequently used to analyze Cryptosporidium isolates from naturally infected calves (n: 123) and lambs (n: 113) from farms in northern Spain. All isolates provided fragments typical of C. parvum. Fragment analysis at the GP60 locus differentiated a total of 10 alleles within isolates from calves (6 alleles) and lambs (8 alleles), with all but three alleles being host-associated. These findings support the validity of the optimized CE approach as a discriminatory and time- and cost-saving alternative to sequencing for identification of Cryptosporidium species and GP60 alleles in domestic ruminants.