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, ¿2), 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 ± 2039 cysts/l and 50 ± 28 cysts/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 ± 105 oocysts/l) and effluent samples (31 ± 70 oocysts/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 20–593 cysts/g and 2–44 oocyst/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 100 l and 125 ± 241 cysts per 100 l. 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 100 l and 37 ± 41 cysts per 100 l, 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

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

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

Prevalencia y grado de parasitación por Syphacia obvelata y Aspicularis tetraptera en ratones NMRI, C57Bl/6 y Balb/c.

Se ha analizado la prevalencia y grado de parasitación p or Syphacia obvelata y Aspicularis tetraptera en ratones de laboratorio, según cepas (NMRI, C57B1/6 y Balb/c), edad, sexo y técnica diagnóstica, seleccionando 300 ratones del Bioterio Central de la Universidad Centroccidental Lisandro Alvarado (Barquisimeto - Venezuela), 100/cepa, 50/sexo, 10/edad, de 3, 5, 7, 9 y 14 semanas. De cada animal se recogieron por duplicado muestras de heces (Técnica de Mc Master), examen directo (heces en ciego) y piel en la región anal (Técnica de celofán). Los datos se analizaron con U de Mann - Whitney, Kruskal - Wallis (P<0, 05 y P<0, 01), correlación de Spearman y análisis multivariable factorial. La prevalencia de S. obvelata en los NMRI (54, 5%) y C57Bl/6 (47, 5%) fue mayor que en Balb/c (19%) así como el grado de parasitación (P<0, 01). Las técnicas de celofán y examen directo en ciego permiten identificar mayor número de animales parasitados que la técnica de Mc Master (P<0, 01). La correlación entre la técnica de celofán y el examen directo es moderada (r=0, 52; P<0, 01) y de ambas té cnicas con Mc Master es baja (r=0, 168; P<0, 01 y r=0, 172; P<0, 01). Existe efecto combinado de cepa, sexo y edad sobre la eliminación de huevos de S. obvelata y presencia de adultos en ciego (P<0, 01). Se identificó A. tetraptera en las tres cepas mediante la técnica de Mc Master y examen directo en ciego, con alta correlación (r=0, 72; P<0, 01) entre las técnicas. Tanto la prevalencia como el grado de parasitación son mayores en los ratones NMRI (67, 5%) que en los C57Bl/6 (2, 5%) y Balb/c (1%) (P<0, 01). Existe influencia del sexo y la edad sobre el recuento de huevos en heces (P<0, 01) y de adultos en ciego (P<0, 05).Se recomienda emplear la técnica de celofán para el diagnóstico de huevos de S. obvelata y tener en cuenta la edad, sexo y cepa de los animales al diseñar los programas de control. The prevalence and intensity of infection with Syphacia obvelata and Aspicularis tetraptera in laboratory mice are reported. The influence of mice strain (NMRI, C57Bl/6 and Balb/c), age and sex on infection were analyzed. The study was conducted in 300 mice randomly selected in the vivarium at the Universidad Centro-occidental Lisandro Alvarado (Barquisimeto, Venezuela).For each strain, a total of 100 mice of different sex and age groups (3, 5, 7, 9 and 14 weeks) were used. The sensitivity of three diagnostic methods was compared, including direct detection in the caecum, analysis of the perianal region by means of cellophane tape and Mc Master egg counting. Both the infection intensity and prevalence of S. obvelata were higher in NMRI (54.5%) and C57Bl/6 (47.5%) mice than in Balb/c mice (19%) (P<0.01). The cellophane tape and direct detection techniques were more sensitive than Mc Master egg counting (P<0.01). The coefficient of ranks of Spearman revealed a moderate correlation between the cellophane tape technique and the direct detection (r = 0.52, P<0.01) and a weak correlation between these techniques and the McMaster egg counting (r = 0.168, P<0.01; r = 0.172, P<0.01, respectively). The multiple factor analysis showed that the presence of adults and eggs of S. obvelata was related to the strain, sex and age of mice (P<0.01). Adults and eggs of A. tetraptera were identified in all the three mice strains using direct detection and McMaster egg counting, with results of both techniques being strongly correlated (r = 0.72; P<0.01). Both the infection intensity and prevalence of A. tetraptera were higher in NMRI (67.5%) mice than in C57Bl/6 (2.5%) and Balb/c mice (1%) (P<0.01).The multiple factor analysis revealed that the presence of adults (P<0.05) and eggs (P<0.01) of A. tetraptera was related to the sex and age of mice. The cellophane tape technique provided the best results to detect eggs of S. obvelata. The strain, sex and age of mice should be taken in consideration to design control programs

Infecciones mixtas por ectoparásitos y endoparásitos en ratones y ratas de laboratorio

Se ha determinado la presencia de parasitosis mixtas en ratones y ratas del Bioterio Central de la Universidad Centroccidental Lisandro Alvarado (UCLA), y la correlación entre ellas, seleccionando 10 animales por sexo, edad (3, 5, 7, 9 y 14 semanas), y cepa de ratón (NMRI, C57Bl/6, Balb/c), y rata (Sprague Dawley y SHR/N) para un total de 500 animales según recomendaciones de Thursfield, (1990). Las muestras se han obtenido por duplicado en vivo y en necropsia de: heces (Examen directo y Mc Master), piel (cuello, dorso-lumbar y ano) con la técnica de celofán; sangre, corazón, pulmón, hígado, bazo, riñones, estómago e intestino (duodeno, yeyuno-íleon y ciego), en frotis teñidos con el método de Giemsa. Se ha determinado la frecuencia de infecciones mixtas y la correlación entre las parasitosis mediante análisis de correlación de Spearman. Las especies identificadas han sido: Myocoptes musculinus, Giardia muris, Trichomona muris, Spironucleus muris, Syphacia obvelata y Apicularis tetraptera en los ratones , y Spironucleus muris solo en NMRI de 6 semanas. En las ratas se ha identificado Radfordia affinis, Giardia muris, Trichomona muris y Syphacia muris. Existe correlación positiva de A. tetraptera con G. muris en ciego (r=0, 492; P<0, 01) y T. muris en duodeno (r=0, 362; P<0, 01). En NMRI casi el 100% están parasitados por al menos 4 especies principalmente machos, éste comportamiento es similar en C57Bl/6 pero con prevalencias inferiores y en los Balb/c predominan 2 ( M. musculinus y G. muris) o 3 especies (M. musculinus , G. muris y T. muris). En ratas se producen infecciones mixtas (99, 9%) por 2-5 especies parásitas. Los flagelados son los más comunes, asociados a S. obvelata o A. tetraptera en ratones, o S. muris en ratas, sumando la presencia de M. musculinus y R. affinis respectivamente. The occurrence of mixed parasitic infections was analyzed in laboratory mice and rats. The study was conducted in 500 animals randomly selected in the vivarium at the Universidad Centro-occidental Lisandro Alvarado (Barquisimeto, Venezuela). A total of 10 animals for each sex (male versus female), age group (3, 5, 7, 9 y 14 weeks) and strain of mice (NMRI, C57Bl/6 and Balb/c) and rats (Sprague Dawley and SHR/N) was selected. Duplicate samples were taken from various locations and analyzed by different methods, including the McMaster technique for faecal egg counting, the cellophane tape technique for skin samples (neck, back, lumbar and anus), and Giemsastaining for smears of different tissues (blood, heart, lung, liver, spleen, kidneys, stomach, duodenum, jejunum, ileum and caecum). Various parasites were identified in mice (Myocoptes musculinus, Giardia muris, Trichomona muris, Spironucleus muris, Syphacia obvelata y Apicularis tetraptera) and rats (Radfordia affinis, Giardia muris, Trichomona muris y Syphacia muris). Spironucleus muris was only identified in NMRI mice aged six weeks. The coefficient of ranks of Spearman revealed a positive correlation between A. tetraptera and G. muris in the caecum (r=0.492; P<0.01), or T. muris in the duodenum (r=0.362; P<0.01). Mixed infections with four species were seen in most NMRI and C57Bl/6 male mice. Mixed infections with two (M. musculinus and G. muris) o three species (M. musculinus, G. muris and T. muris) predominated in Balb/c mice. Mixed infections with two-to-five species were seen in most rats. Flagellate protozoa were the most prevalent parasites in both mice and rats, followed by S. obvelata and A. tetraptera (mice) or S. muris (rats)

Prevalencia y grado de parasitación por flagelados entéricos en ratones NMRI, C57Bl/6 y Balb/c

In order to analyze the prevalence and parasitation degree by enteric flagellates in NMRI, C57Bl/6 and Balb/c mice, discriminating on the basis of strain, age and sex of animals produced conventionally in animal facilities of the Universidad Centroccidental Lisandro Alvarado (Venezuela), 50 mice were selected by strain and sex, and 10 by age (3, 5, 7, 9 and 14 weeks) (N = 300). They were sampled both fresh feces and intestinal contents at three differents sections (duodenum, yeyuno-ileon and cecum), for direct observation and smears stained with Giemsa. The data was analyzed with Chi-square, Mann-Whitney, Kruskal-Wallis test, correlation of Spearman and factor analysis. Giardia muris was located at cecum of NMRI mice (100%) and 99% of C57Bl/6 and Balb/c. At yeyuno-ileon in 89% of C57Bl/6, 84% of NMRI and 46% of Balb/c; and at duodenum, in male C57Bl/6 (73%), which increases the prevalence with age. Trichomonas muris was identified in 58-76% of the three strains with the highest prevalence in males NMRI and C57B1/6. Spironucleus muris, only 70% of the six-weeks-old NMRI were identified. The less parasitized strain is the Balb/c. There are significant differences in the prevalence of three parasitic diseases among the strains studied, about Giardia there are differences between the ages in the NMRI and C57Bl/6 and about Trichomonas between both sex. It is recommended to consider animals strain produced and/or used in research, their sex and age in order to establish the sanitary controls aimed to enteric flagellates.Con el objetivo de analizar la prevalencia y grado de parasitación por flagelados entéricos en ratones NMRI, C57Bl/6 y Balb/c, discriminando en base a la cepa, edad y sexo de los animales producidos convencionalmente en el Bioterio de la Universidad Centroccidental Lisandro Alvarado (Venezuela), se seleccionaron 50 ratones por cada cepa y sexo, 10 por edad (3, 5, 7, 9 y 14 semanas) (N=300). Se recogieron por duplicado muestras de heces frescas y de contenido intestinal en tres tramos diferentes (duodeno, yeyuno-íleon y ciego), para observación directa y frotis teñido con Giemsa. Los datos fueron analizados con Chi-cuadrado, U de Mann-Whitney, Prueba de Kruskal-Wallis, correlación de Spearman y análisis factorial. Giardia muris se localizó en ciego de los ratones NMRI (100%) y en 99% de los C57Bl/6 y Balb/c. En yeyuno-íleon en 89% de los C57Bl/6, 84% de NMRI y 46% de Balb/c; y en duodeno, en los machos C57Bl/6 (73%), en los que aumenta la prevalencia con la edad. Se identificó Trichomonas muris en 58-76% de las tres cepas con la máxima prevalencia en machos NMRI y C57Bl/6. Spironucleus muris, solo se identificó en 70% de los NMRI de 6 semanas. La cepa menos parasitada es la Balb/c. Existen diferencias significativas en la prevalencia de las tres parasitosis entre las cepas estudiadas, en Giardia diferencias entre las edades en NMRI y C57Bl/6 y en Trichomonas entre ambos sexos. Existen diferencias significativas en la prevalencia de las tres parasitosis entre las cepas estudiadas, en Giardia diferencias entre las edades en NMRI y C57Bl/6 y en Trichomonas entre ambos sexos. Se recomienda tomar en cuenta la cepa de los animales producidos y/o empleados en investigación, su sexo y edad para establecer los controles sanitarios dirigidos a flagelados entéricos

The current importance of Leishmaniasis in Spain

Se pasa revisión al estado actual de los principales elementos epidemiológicos de las leishmaniosis en España: parásito, reservorio canino, flebotomo vector, y hombre, poniendo de manifiesto la importancia sanitaria, social y económica de la enfermedad. Se termina recogiendo datos recientes sobre el diagnóstico y tratamiento de la enfermedad.A revision is made of the current state of the principal epidemiological elements of leishmaniasis in Spain (the parasite, the canine population, the sandflies vector, and humans), while stating the sanitary, social and economic significance of the disease. Finally, recent information is given regarding the diagnosis and treatment of the disease