IIa Subtype Research Articles

Diagnosis and classification of Mirizzi syndrome

Objective. To determine the factors, predisposing to development of Mirizzi syndrome; to improve diagnosis and classification of it, taking into account of these factors and possibilities to apply a definite kind of endoscopic or operative treatment.
 Materials and methods. Retrospective analysis was done for results of treatment of 21 patients with Mirizzi syndrome: Type I in accordance to classification of McSherry (1982) was present in 7 patients, while a Type II - in 14.
 Results. There was established, that atypia (the variant anatomy) of the ductus cysticus localization predisposes for contact between hepaticocholedochus and a gallbladder of with ductus cysticus, leading to development of Mirizzi syndrome Types I and II. Squeezing (Type I) or fistula (Type II) are observed on any level of hepaticocholedochus. In syndrome of Mirizzi Type II the duct distal to fistula may be not dilated (Subtype IIA) or dilated (Subtype IIB).
 Conclusion. Atypia (the variant anatomy) of the ductus cysticus duct constitute a factor, promoting development of Mirizzi syndrome. While diagnostic process for Mirizzi syndrome it is necessary to take into account a localization of squeezing of hepaticocholedochus or of fistula, presence of atypia of the ductus cysticus and its stump localization, character of the gallbladder inflammation, quantity and size of calculi. Proposition to include the Types IIA and IIB II in Mirizzi syndrome, depending on the dilation degree present in hepaticocholedochus distally, to the fistula, constitutes a substantiated principle, because it takes into account a possibility to perform endoscopic lithoextraction and to impact the choice of the surgical treatment method.

Positive EGFR mutation status is a risk of recurrence in pN0–1 lung adenocarcinoma when combined with pathological stage and histological subtype: A retrospective multi-center analysis

Objectives: Recurrence risk of resected lung adenocarcinoma is represented by pathological stage (pStage), histological subtype, and potentially by EGFR mutation. However, the relationship among these factors and their combined impact on prognosis are unclear. Materials and MethodsUsing a multicenter database, we retrospectively investigated the prognostic impact of EGFR mutation status in relation to pStage and histological subtype in resected pN0–1M0 lung adenocarcinoma. ResultsAmong 1155 pN0–1M0 adenocarcinoma cases, pStage 0 and IA1–IB were confirmed predominantly in EGFR-positive cases. AIS, MIA, and lepidic predominant adenocarcinoma were also more frequently found in EGFR-positive cases and showed no/little recurrence regardless of EGFR mutation status. The 5-year recurrence-free survival (RFS) of papillary, acinar, solid, and micropapillary predominant adenocarcinoma was stratified by pStage (IA1–IB, IIA–IIIA) or histological malignant subtype (intermediate or high malignant subtype), and more finely subdivided by EGFR mutation status. Positive EGFR mutation cases showed worse RFS in both classifications. Low malignant subtype and pStage IA1–IB intermediate malignant subtype showed low frequency of recurrence. Whereas, in pStage IA1–IB high malignant subtype and pStage IIA–IIIA cases, EGFR-positive cases showed poorer 5-year RFS than EGFR-negative (49.6% and 75.6%, respectively, hazard ratio [HR] = 1.84, 95% CI = 1.38–7.42, p < 0.01) and multivariate analysis indicated positive EGFR mutation status was significantly related to poorer PRF (HR = 2.005, 95% CI = 1.029–3.906, p = 0.041). ConclusionEGFR mutation harbored primarily in early-stage or low-malignant histological subtypes with no/little recurrence. In pN0–1M0 adenocarcinoma with higher risk of recurrence, positive EGFR mutation cases showed worse RFS. EGFR mutation status enables better stratification of recurrence risk when considering pStage and histological malignant subtype.

Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches.

Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

First identification of Cryptosporidium parvum zoonotic subtype IIaA15G2R1 in diarrheal lambs in France.

To date, no information is available about the presence of Cryptosporidium spp. in French sheep, nor their potential role as zoonotic reservoirs. A total of 23 fecal samples were collected from diarrheic lambs (<11days old) from seven randomly selected farms. Cryptosporidium-oocysts were detected microscopically with Direct Immunofluorescence Assays (DFA) in 23/23 (100%) of fecal samples. PCR-RFLP of the 18S rRNA gene was used to determine species in all samples, and only Cryptosporidium parvum was identified. Isolates were subtyped by sequencing the 60kDa glycoprotein (gp60) gene. Two zoonotic subtypes within the IIa subtype family were identified, including IIaA15G2R1 (22/23) and IIaA16G3R1 (1/23). This study reports for the first time the identification and genotyping of zoonotic C. parvum subtypes from lambs in France. Sheep could thus play an important role as potential reservoirs for this zoonotic protist.

391O - Prognostic role of the EANO ESMO classification of leptomeningeal metastases

BackgroundThe EANO ESMO guidelines have proposed a classification of leptomeningeal metastases (LM) based on clinical (typical/atypical), cytological (positive/negative/equivocal) and MRI (A linear, B nodular, C linear + nodular, D normal or hydrocephalus only) presentation. Type I LM is defined by the presence of tumor cells in the cerebrospinal fluid (CSF) (confirmed LM) whereas type II LM is defined by typical clinical and MRI signs (probable or possible LM). Here we explored the correlation between EANO ESMO subtypes of LM and outcome to assess their clinical utility. MethodsWe collected data from 254 patients with newly diagnosed LM from different primary solid tumors. Survival curves were estimated using the Kaplan-Meier method and compared by Log-rank test. ResultsMedian age at LM diagnosis was 56.5 years (range 20-82 years). The main primary tumors were breast cancer (n=98, 45%), lung cancer (n=65, 25.5%) and melanoma (n=51, 13.5%). Typical clinical LM symptoms or signs were noted in 225 patients (88.5%); only 13 patients (5%) were clinically asymptomatic. The most common MRI subtype was A seen in 117 patients (46%). Types B (n=33, 13%), C (n=54, 21%) and D (n=50, 19.5%) were less common. Tumor cells were observed in the CSF in 186 patients (73%) whereas the CSF was equivocal in 24 (9.5%) and negative in 44 (17.5%) patients. Assignment to EANO ESMO classes was as follows: type IA 86 (34%), IB 19 (7.5%), IC 39 (15.5%), ID 45 (17.5%), IIA 28 (11%), IIB 13 (5%), IIC 16 (6.5%), IID 8 (3%). LM was confirmed in 189 (74.5%), probable in 51 (20%) and possible in 14 (5.5%) patients. The median overall survival (OS) was 2.75 months (range 0.09-220 months). OS was inferior in type I compared with type II LM patients (p=0.0024). EANO ESMO subtype IIA patients had the longest survival (p=0.0103). Patients with MRI patterns A and D pooled had poorer survival than B and C patients pooled in the presence of tumor cells in the CSF (type I) (p=0.0402), but longer survival in the absence of tumor cells in the CSF (type II) (p=0.0273). ConclusionsThe presence of tumor cells in the CSF appears to have a greater prognostic role than the neuroimaging presentation. EANO ESMO LM subtypes are prognostic and should be considered in the design of clinical trials. Legal entity responsible for the studyUniversity Hospital of Zurich. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Divergent Copies of a Cryptosporidium parvum-Specific Subtelomeric Gene.

Subtype families of Cryptosporidium parvum differ in host range, with IIa and IId being found in a broad range of animals, IIc in humans, and IIo and IIp in some rodents. Previous studies indicated that the subtelomeric cgd6_5520-5510 gene in C. parvum is lost in many Cryptosporidium species, and could potentially contribute to the broad host range of the former. In this study, we identified the presence of a second copy of the gene in some C. parvum subtype families with a broad host range, and showed sequence differences among them. The sequence differences in the cgd6_5520-5510 gene were not segregated by the sequence type of the 60 kDa glycoprotein gene. Genetic recombination appeared to have played a role in generating divergent nucleotide sequences between copies and among subtype families. These data support the previous conclusion on the potential involvement of the insulinase-like protease encoded by the subtelomeric cgd6_5520-5510 gene in the broad host range of C. parvum IIa and IId subtypes.

Relationship between the characteristics of rigor-mortis-related actomyosin and muscle fiber types in the ordinary muscle of various fishes.

Rigor mortis occurs when muscle extension vanishes through the irresistible coupling of actin and myosin by the consumption of adenosine triphosphate as energy. To clarify the cause of the differences in the progression of rigor mortis, seven fish species were used as samples. The superprecipitation reaction and Mg2+ -ATPase activity of actomyosin in dorsal ordinary muscle were measured, and the slope of the regression line between these two variables was calculated for each fish specimen. The fiber types of the dorsal ordinary muscle in each sample fish were discriminated by the stability of actomyosin ATPase at acid and alkaline preincubations. Positive correlations were found between Mg2+ -ATPase activity and the superprecipitation reaction of actomyosin in all 27 fish specimens. The slopes of the regression lines were different not only between fish species but also in fish specimens within the same species. The area ratios of pink muscle fibers and the IIa and/or IIb subtypes of white muscle fibers in the dorsal ordinary muscle were also different between fish species, as well as in specimens within the same fish species. A positive correlation was found between the area ratios of pink muscle fibers in dorsal ordinary muscle and the slopes of the regression line. It was suggested that the differences in characteristics of rigor-mortis-related actomyosin of fish might have been caused by the differences in the interposition ratio of muscle fiber types, especially of the pink muscle fiber type, in the dorsal ordinary muscle. © 2019 Society of Chemical Industry.

Molecular characterization of Cryptosporidium isolates from diarrheal dairy calves in France.

Cryptosporidium is an obligate intracellular protist parasite infecting a wide range of vertebrate hosts and causes significant intestinal disease in both animals and humans, as some species are zoonotic. Cattle and especially calves have been identified as one of the most common reservoirs of this protist. However, little is known about the genetics of Cryptosporidium in calves in some regions of France. The aim of this study was to detect and isolate Cryptosporidium spp. in faecal samples from naturally infected pre-weaned calves (≤45 days-old) in France. A total of 35 diarrhoeic pre-weaned calf faecal samples were collected from 26 dairy cattle farms in six departments (French administrative provinces). Cryptosporidium presence was established by microscopically screening samples for oocystes with an immunofluorescent (DFA) staining method. DFA-positive samples were then analysed by PCR-RFLP and 18S rRNA gene sequencing to determine species. Cryptosporidium parvum-positive samples were subtyped via nested PCR analysis of a partial fragment of the 60 kDa glycoprotein (gp60) gene product. Data were then integrated into phylogenetic tree analysis. DFA revealed the presence of Cryptosporidium oocysts in 31 out of 35 (88%) samples. Combined with 18S rRNA gene analysis results, C. parvum was detected in 30 samples. Subtyping analysis in 27/30 samples (90%) of the C. parvum isolates revealed two zoonotic subtype families, IIa (24/27) and IId (3/27). Four subtypes were recognised within the subtype family IIa, including the hypertransmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (21/27), IIaA17G3R1 (1/27), IIaA17G1R1 (1/27), and IIaA19G1R1 (1/27). Two subtypes were recognised within the IId subtype family including IIdA22G1 (2/27) and IIdA27G1 (1/27). These findings illustrate the high occurrence of Cryptosporidium in calves in dairy herds and increase the diversity of molecularly characterised C. parvum isolates with the first description of IIaA17G3R1, IIaA19G1R1, and IId subtypes in France. The presence of zoonotic C. parvum subtype families (IIa, IId) in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, and highlights the importance of animal to human cryptosporidiosis transmission risk. Further molecular studies in calves and small ruminants from other French regions are required to better understand the epidemiology of cryptosporidiosis in France.

Diversity of CRISPR/Cas system in Clostridium perfringens.

Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.

Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep.

Little is known about the presence of Cryptosporidium spp. and Giardia duodenalis in Algerian sheep, nor their potential role as zoonotic reservoirs. This study aimed to investigate the occurrence and distribution of these two protists in lambs. A total of 83 fecal samples were collected from lambs (< 40 days old) from 14 different farms. Samples were screened for Cryptosporidium spp. and Giardia duodenalis presence using immunofluorescent techniques (IF). Nested PCR of the small subunit ribosomal RNA (rRNA) gene, followed by restriction fragment length polymorphism (PCR-RFLP) and sequence analyses were used to identify Cryptosporidium species. C. parvum was further subtyped by sequencing the highly polymorphic 60 kDa glycoprotein (gp60) gene. For G. duodenalis, nested PCR of the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes was performed and then PCR-RFLP was used to identify G. duodenalis assemblages. Cryptosporidium oocysts and Giardia cysts were detected in 36/83 (43%) and 23/83 (28%) of fecal samples, respectively. Of the 21/36 (58%) Cryptosporidium samples that were positive with IF, 16/21 (76%) were identified as C. parvum, and 5/21 (24%) as C. ubiquitum. From 15C. parvum isolates, 2 subtypes were identified within the IIa subtype family, including IIaA21G2R1 (3/15) and IIaA13G2R1 (1/15), while IIdA16G1 (11/15) was the only subtype identified from the IId subtype family. Of the 16/23 (69%) G. duodenalis IF-positive samples, the most frequent assemblage was ruminant-specific assemblage E (10/16), followed by assemblage D (4/16), and A + E mixed assemblages (2/16). This study is the first to identify and genotype both Cryptosporidium spp. and Giardia duodenalis in Algerian lambs, and is also the first to describe G. duodenalis assemblage D in small ruminants. The presence of zoonotic C. parvum subtype families (IIa, IId), C. ubiquitum, as well as G. duodenalis assemblage A + E, indicates that sheep could play an important role as a potential reservoir for protists.

Molecular and descriptive epidemiology of intestinal protozoan parasites of children and their pets in Cauca, Colombia: a cross-sectional study

BackgroundParasitic infections, particularly those caused by protozoa, represent a considerable public health problem in developing countries. Blastocystis, Giardia duodenalis, Cryptosporidium spp. and the Entamoeba complex (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii) are the most common etiological causes of intestinal parasitic infections.MethodsWe carried out a descriptive cross-sectional study in school-age children attending a daycare institution in commune eight of Popayán, Cauca (Southwest Colombia). A total of 266 fecal samples were collected (258 from children and eight from pets). Blastocystis, G. duodenalis, Cryptosporidium spp. and the Entamoeba complex were identified by microscopy, quantitative real-time PCR (qPCR) and conventional PCR. The concordance of qPCR and microscopy was assessed using the Kappa index. Molecular characterization was conducted to identify Blastocystis subtypes (18S), G. duodenalis assemblages (tpi and gdh) and Cryptosporidium species/subtypes (18S and GP60). Potential associations between intestinal parasitism and sociodemographic factors were examined using bivariate analyses.ResultsA total of 258 fecal samples from children were analyzed by microscopy and 255 samples were analyzed by qPCR. The prevalence of Blastocystis was between 25.19% (microscopy) and 39.22% (qPCR), that of G. duodenalis was between 8.14% (microscopy) and 10.59% (qPCR), that of Cryptosporidium spp. was estimated at 9.8% (qPCR), and that of the Entamoeba complex was between 0.39% (conventional PCR) and 0.78% (microscopy). The concordance between microscopy and qPCR was very low. Blastocystis ST1 (alleles 4, 8, and 80), ST2 (alleles 11, 12, and 15), ST3 (alleles 31, 34, 36, 38,57, and 151), and ST4 (alleles 42 and 91), G. duodenalis assemblages AII, BIII, BIV and D, C. parvum subtype IIa and C. hominis subtype IbA9G3R2 were identified. The only identified member of the Entamoeba complex corresponded to E. histolytica. No statistically significant association was identified between parasitic infection and any sociodemographic variable.ConclusionThis study revealed the usefulness of molecular methods to depict the transmission dynamics of parasitic protozoa in southwest Colombia. The presence of some of these protozoa in domestic animals may be involved in their transmission.

Genotypes and subtypes of Cryptosporidium spp. in diarrheic lambs and goat kids in northern Greece

Inconsistent data exist on the distribution of zoonotic Cryptosporidium species and subtypes in sheep and goats in European countries, and few such data are available from Greece. In this study, 280 fecal specimens were collected from 132 diarrheic lambs and 148 diarrheic goat kids aged 4 to 15 days on 15 farms in northern Greece, and examined for Cryptosporidium spp. using microscopy of Ziehl-Neelsen-stained fecal smears. Cryptosporidium spp. in 80 microscopy-positive fecal specimens (39 from lambs and 41 from goat kids) were genotyped by PCR-RFLP analysis of the small subunit rRNA gene and subtyped by sequence analysis the 60 kDa glycoprotein gene. Among the 33 specimens successfully genotyped, C. parvum was found in 32 and C. xiaoi in one. Seven subtypes belonging to two subtype families (IIa and IId) were identified among the 29 C. parvum specimens successfully subtyped, including IIaA14G2R1 (1/29), IIaA15G2R1 (6/29), IIaA20G1R1 (7/29), IIdA14G2 (1/29), IIdA15G1 (9/29), IIdA16G1 (3/29), and IIdA23G1 (2/29). Lambs were more commonly infected with C. parvum IIa subtypes, whereas goat kids were more with IId subtypes. The results illustrate that C. parvum is prevalent in diarrheic lambs and goat kids in northern Greece and these animals could potentially play a role in epidemiology of human cryptosporidiosis.

Proposal for a new classification of the renal artery in the bovine kidney

Proper vascularisation is necessary for the correct functioning of all organs. The kidneys of various mammalian species have been examined in order to understand the functioning of this organ. This article presents the first classification of the renal artery division in the kidneys of adult cattle. We collected and analysed specimens of arteries from bovine kidneys with the aim of improving our understanding of their morphology and functioning. The study was conducted on 50 kidneys, 25 right ones and 25 left ones, taken from cattle of both sexes. The examined kidneys were dissected and corrosion casts were made. Division of the renal artery into between two and four primary segmental arteries takes place just before entering the renal hilum. Cranial primary segmental arteries number from one to two (most frequently one), whereas the hilar and caudal ones always occur singly. The mentioned vessels are then divided into between one and four secondary segmental arteries running within the renal sinus. The hilar region (mid-zone) of the kidney exhibits the most variation in terms of vascularisation. The vascularisation of the caudal pole exhibits the lowest degree of variation. Taking into consideration the range of vascularisation of the organ by the particular divisions of the renal artery, three renal branching pattern types were distinguished: type I (84.1% of cases; this type consists of Ia, Ib, Ic and Ic subtypes) – two branches – the renal artery is divided into cranial and caudal primary segmental arteries; type II (11.36% of cases; with IIa and IIb subtypes) – three branches – the renal artery is divided into the cranial, hilar and caudal primary segmental arteries; type III (4.54% of cases) – four branches – the renal artery is divided into two cranial, one hilar and one caudal primary segmental artery. The division of the renal artery takes place along the long axis of the organ. Bovine kidneys are characterised by asymmetry, which may influence the length and diameter of the main arteries. The caudal primary segmental artery has the biggest mean length and mean diameter. The division of the renal artery occurs just before it enters the renal hilum or in the renal sinus.

RNA-dependent RNA targeting by CRISPR-Cas9.

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

Advances and Perspectives on the Epidemiology of Bovine Cryptosporidium in China in the Past 30 Years.

Major progress has been made in understanding the epidemiology of bovine Cryptosporidium in China in the past 30 years. The overall infection rate in that period was 14.50% (5265/36316), with different prevalence being observed among dairy cattle, yaks, beef cattle, and buffalo. The infection rate declined as the animals’ ages increased and the lowest prevalence occurred in winter. Ten Cryptosporidium species and two genotypes have been found in cattle, with Cryptosporidium parvum, C. andersoni, C. bovis, and C. ryanae being the commonest species. Cryptosporidium bovis rather than C. parvum predominated in preweaned dairy cattle, and C. parvum IIdA15G1 and IIdA19G1 were the only subtypes detected in dairy cattle. Two subtype families, IIa and IId, were found in yaks. Population genetic analysis detected an epidemic population structure in C. andersoni, which suggested that the prevalence of C. andersoni in China is not attributable to the introduction of dairy cattle. Moreover, C. parvum IId subtypes probably dispersed from western Asia to other geographic regions based on population genetic analysis of isolates from China, Sweden, and Egypt. Therefore, we hypothesize that Cryptosporidium was introduced into China in the past, and different populations formed progressively in various hosts in response to diverse factors, including the transmission dynamics, geographic isolation, host specificity, and large-scale farming. More epidemiological studies are required to test this hypothesis and to clarify the prevalence and transmission of Cryptosporidium species in China.

Molecular Epidemiology of Cryptosporidiosis in China.

Molecular epidemiology of cryptosporidiosis is an active research area in China. The use of genotyping and subtyping tools in prevalence studies has led to the identification of unique characteristics of Cryptosporidium infections in humans and animals. Human cryptosporidiosis in China is exemplified by the high diversity of Cryptosporidium spp. at species and subtype levels, with dominant C. hominis and C. parvum subtypes being rarely detected in other countries. Similarly, preweaned dairy calves, lambs, and goat kids are mostly infected with non-pathogenic Cryptosporidium species (C. bovis in calves and C. xiaoi in lambs and goat kids), with C. parvum starting to appear in dairy calves as a consequence of concentrated animal feeding operations. The latter Cryptosporidium species is dominated by IId subtypes, with IIa subtypes largely absent from the country. Unlike elsewhere, rodents in China appear to be commonly infected with C. parvum IId subtypes, with identical subtypes being found in these animals, calves, other livestock, and humans. In addition to cattle, pigs and chickens appear to be significant contributors to Cryptosporidium contamination in drinking water sources, as reflected by the frequent detection of C. suis, C. baileyi, and C. meleagridis in water samples. Chinese scientists have also made significant contributions to the development of new molecular epidemiological tools for Cryptosporidium spp. and improvements in our understanding of the mechanism involved in the emergence of hyper-transmissible and virulent C. hominis and C. parvum subtypes. Despite this progress, coordinated research efforts should be made to address changes in Cryptosporidium transmission because of rapid economic development in China and to prevent the introduction and spread of virulent and zoonotic Cryptosporidium species and subtypes in farm animals.

Spines vs. microspines: an overview of the sculpture exine in selected basal and derived Asteraceae with focus on Asteroideae.

This study presents a detailed examination of the echinate and microechinate sculpturing in relation to the size of pollen grains in 31 selected species of Asteraceae belonging to the subfamilies Barnadesioideae, Mutisioideae, Carduoideae and Asteroideae. The aims were to recognize sculpturing patterns, under LM and SEM, within large and small pollen of both basal and derived species and to explore the features that could have taxonomic value to apply in palynological disciplines. The detailed examination of the exine surface showed both the relevance and limits of sculptural patterns for taxonomy. Under LM, the microechinate sculpture gave little taxonomic information, whereas in the echinate sculpture, three exine types and two subtypes were recognized. Type I included microechinate exine, which is commonly present in large pollen grains of the basal lineages. Types II (subtypes IIa and IIb) and III included echinate and smaller pollen grains. In these types, spines were always regularly arranged and, were characterized by the length, shape, tip, perforations and distribution. Type IIa included more or less conical spines usually with a distended base, less than 4µm in length, present in species of different tribes like Astereae, Eupatorieae, Helenieae, Gnaphalieae, Senecioideae and Heliantheae to a lesser extent. Type IIb includes slender spines with narrower bases, longer than 4µm, present in species of Coreopsideae, Heliantheae, Tageteae and Eupatorieae to a lesser extent. Type III included spines with swollen base, blunt tip and perforations over their entire surface. This type was present in only one of the basal species-Carduus thoermeri-and in one species of the derived tribe Helenieae, Gaillardia megapotamica. Probably, this is due to evolutionary convergence.

Next Generation Sequencing uncovers within-host differences in the genetic diversity of Cryptosporidium gp60 subtypes

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, disease severity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing. The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n=11), Cryptosporidium parvum (n=22) and Cryptosporidium cuniculus (n=8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n=11), four C. parvum subtypes belonging to IIa (n=3) and IId (n=19) and one C. cuniculus subtype (VbA23) (n=8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies.

Comparative genomic analysis of the IId subtype family of Cryptosporidium parvum

Host adaptation is known to occur in Cryptosporidium parvum, with IIa and IId subtype families preferentially infecting calves and lambs, respectively. To improve our understanding of the genetic basis of host adaptation in Cryptosporidium parvum, we sequenced the genomes of two IId specimens and one IIa specimen from China and Egypt using the Illumina technique and compared them with the published IIa IOWA genome. Sequence data were obtained for >99.3% of the expected genome. Comparative genomic analysis identified differences in numbers of three subtelomeric gene families between sequenced genomes and the reference genome, including those encoding SKSR secretory proteins, the MEDLE family of secretory proteins, and insulinase-like proteases. These gene gains and losses compared with the reference genome were confirmed by PCR analysis. Altogether, 5,191–5,766 single nucleotide variants were seen between genomes sequenced in this study and the reference genome, with most SNVs occurring in subtelomeric regions of chromosomes 1, 4, and 6. The most highly polymorphic genes between IIa and IId encode mainly invasion-associated and immunodominant mucin proteins, and other families of secretory proteins. Further studies are needed to verify the biological significance of these genomic differences.