Sheather's Sugar Flotation Technique Research Articles

INVESTIGATION ON CRYPTOSPORIDIUM INFECTIONS IN WILD ANIMALS IN A ZOO IN ANHUI PROVINCE.

To assess Cryptosporidium infections among wild animals in a zoo located in Anhui province, we conducted an investigation on the fecal samples collected from 44 primates, 41 herbivores, 44 carnivores and omnivores, and 103 birds in the zoo with the use of Sheather's sugar flotation technique and modified acid-fast staining. Cryptosporidium oocysts were detected in the fecal samples from six primates, two herbivores, four carnivores and omnivores, and seven birds by using Sheather's sugar flotation technique; the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 13.64, 4.88, 9.09, and 6.80%, respectively. Modified acid-fast staining detected the presence of Cryptosporidium oocysts in the fecal samples of one primate, three herbivores, 0 carnivores and omnivores, and one bird, and the prevalence of Cryptosporidium infection in primates, herbivores, carnivores and omnivores and birds was 2.27, 7.32, 0.00, and 0.97%, respectively. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) and phylogenetic analysis with the use of the neighbor-joining (NJ) method based on the aligned partial small-subunit (SSU) rRNA gene sequences showed that the protozoan pathogen isolated from primates was Cryptosporidium hominis and the pathogen isolated from camels ( Camelus dromedarius ) was Cryptosporidium andersoni. Subtyping the Cryptosporidium hominis by 60-kDa glycoprotein (GP60) gene phylogenetic analysis showed the Cryptosporidium hominis belongs to the subtype IdA and IbA.

Prevalence and multilocus genotyping of Cryptosporidium andersoni in dairy cattle and He cattle in Xinjiang, China

Cryptosporidium andersoni is the predominant species in post-weaned and adult cattle in China. The aim of this study was to determine the prevalence and understand the transmission of cattle cryptosporidiosis in the Xinjiang Uyghur Autonomous Region, China, a total of 1827 fecal samples (436 from He cattle and 1391 from dairy cattle) were examined for the presence of C. andersoni-like oocysts by microscopy after Sheather's sugar flotation technique. The overall prevalence of C. andersoni-like was 3.8% (70/1827) and all the C. andersoni-like isolates were identified as C. andersoni at the SSU rRNA locus. Among the C. andersoni isolates, a total of 60 isolates were successfully characterized into eight multilocus sequence typing (MLST) subtypes using MLST analysis at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16), and three new subtypes were identified. The MLST subtype A4,A4,A4,A1 showed a predominance and a wide distribution among the eight MLST subtypes obtained in the investigated areas. The MLST subtypes A2,A4,A2,A1 and A4,A5,A2,A1 showed a unique distribution in the investigated areas. A linkage disequilibrium analysis showed the presence of an epidemic population genetic structure of C. andersoni isolated from dairy and He cattle in Xinjiang. These findings provide new insights into the genetic structure of C. andersoni isolates and are also helpful to explore the infection source of C. andersoni in cattle in Xinjiang, China.

The first report of Cryptosporidium andersoni in horses with diarrhea and multilocus subtype analysis.

BackgroundHorses interact with humans in a wide variety of sport competitions and non-competitive recreational pursuits as well as in working activities. Cryptosporidium spp are one of the most important zoonotic pathogens causing diarrhea of humans and animals. The reports of Cryptosporidium in horses and the findings of zoonotic Cryptosporidium species/genotypes show a necessity to carry out molecular identification of Cryptosporidium in horses, especially in diarrheic ones. The aim of the present study was to understand Cryptosporidium infection and species/genotypes in diarrheic horses, and to trace the source of infection of horse-derived Cryptosporidium isolates at a subtype level.FindingsFecal specimens of 29 diarrheic adult horses were collected in Taikang County in northeastern China’s Heilongjiang Province. Cryptosporidium oocysts were concentrated by Sheather’s sugar flotation technique, and then examined by a bright-field microscope. Meanwhile, all the specimens were subjected to PCR amplification of the small subunit (SSU) rRNA gene of Cryptosporidium. C. andersoni isolates were further subtyped by multilocus sequence typing (MLST) at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16).One and two Cryptosporidium-positive isolates were obtained in horses by microscopy and by PCR, respectively. The two C. andersoni isolates were identified by sequencing of the SSU rRNA gene of Cryptosporidium. Both of them were identical to each other at the MS1, MS2, MS3 and MS16 loci, and MLST subtype A4,A4,A4,A1 was found here.ConclusionsThis is the first report of C. andersoni in horses. The fact that the MLST subtype A4,A4,A4,A1 was reported in cattle suggests a large possibility of transmission of C. andersoni between cattle and horses.

Molecular characterization of Cryptosporidium spp. in domestic pigeons (Columba livia domestica) in Guangdong Province, Southern China.

To investigate the prevalence and assess the zoonotic transmission burden of Cryptosporidium species in domestic pigeons in Guangdong Province, Southern China, 244 fecal samples were collected from four pigeon breeding farms between June 2012 and March 2013. Cryptosporidium oocysts were purified by Sheather's sugar flotation technique and characterized by DNA sequencing of small subunit ribosomal RNA (SSU rRNA) gene. Cryptosporidium species were determined by comparison of sequences with corresponding Cryptosporidium sequences in GenBank and phylogenetic analysis using neighbor-joining (NJ) in MEGA5.2. The overall prevalence of Cryptosporidium infection in domestic pigeons in Guangdong Province was 0.82% (2/244). Two Cryptosporidium species, namely Cryptosporidium baileyi and Cryptosporidium meleagridis, were identified in Huizhou and Chaozhou farm, respectively. These findings confirmed the existence of C. meleagridis infection in domestic pigeons in China for the first time and provided base-line information for further studies to evaluate the public health risk from pigeon to human.

MLST subtypes and population genetic structure of Cryptosporidium andersoni from dairy cattle and beef cattle in northeastern China's Heilongjiang Province.

Cattle are the main reservoir host of C. andersoni, which shows a predominance in yearlings and adults of cattle. To understand the subtypes of C. andersoni and the population genetic structure in Heilongjiang Province, fecal specimens were collected from 420 dairy cattle and 405 beef cattle at the age of 12–14 months in eight cattle farms in five areas within this province and were screened for the presence of Cryptosporidium oocysts by microscopy after Sheather’s sugar flotation technique. The average prevalence of Cryptosporidium spp. was 19.15% (158/825) and all the Cryptosporidium isolates were identified as C. andersoni by the SSU rRNA gene nested PCR-RFLP using SspI, VspI and MboII restriction enzymes. A total of 50 C. andersoni isolates were randomly selected and sequenced to confirm the RFLP results before they were subtyped by multilocus sequence typing (MLST) at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16). Four, one, two and one haplotypes were obtained at the four loci, respectively. The MLST subtype A4,A4,A4,A1 showed an absolute predominance and a wide distribution among the six MLST subtypes obtained in the investigated areas. Linkage disequilibrium analysis showed the presence of a clonal population genetic structure of C. andersoni in cattle, suggesting the absence of recombination among lineages. The finding of a clonal population genetic structure indicated that the prevalence of C. andersoni in cattle in Heilongjiang Province is not attributed to the introduction of cattle. Thus, prevention and control strategies should be focused on making stricter measures to avoid the occurrence of cross-transmission and re-infection between cattle individuals. These molecular data will also be helpful to explore the source attribution of infection/contamination of C. andersoni and to elucidate its transmission dynamics in Heilongjiang Province, even in China.

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.

Cryptosporidium cuniculus and Giardia duodenalis in Rabbits: Genetic Diversity and Possible Zoonotic Transmission

Background Cryptosporidium and Giardia are the two important zoonotic pathogens causing diarrhea of humans and animals worldwide. Considering the human cryptosporidiosis outbreak and sporadic cases caused by C. cuniculus, the important public health significance of G. duodenalis and little obtained information regarding rabbit infected with Cryptosporidium and Giardia in China, the aim of this study is to determine the prevalence and molecularly characterize Cryptosporidium and Giardia in rabbits in Heilongjiang Province, China.Methodology/Principal Findings378 fecal samples were obtained from rabbits in Heilongjiang Province. Cryptosporidium oocysts and Giardia cysts were detected using Sheather's sugar flotation technique and Lugol's iodine stain method, respectively. The infection rates of Cryptosporidium and Giardia were 2.38% (9/378) and 7.41% (28/378), respectively. Genotyping of Cryptosporidium spp. was done by DNA sequencing of the small subunit rRNA (SSU rRNA) gene and all the nine isolates were identified as Cryptosporidium cuniculus. The nine isolates were further subtyped using the 60-kDa glycoprotein (gp60) gene and two subtypes were detected, including VbA32 (n = 3) and a new subtype VbA21 (n = 6). G. duodenalis genotypes and subtypes were identified by sequence analysis of the triosephosphate isomerase (TPI) gene. The assemblage B (belonging to eight different subtypes B-I to B-VIII) was found in 28 G. duodenalis-positive samples.Conclusions/SignificanceThe rabbits have been infected with Cryptosporidium and Giardia in Heilongjiang Province. The results show that the rabbits pose a threat to human health in the studied areas. Genotypes and subgenotypes of C. cuniculus and G. duodenalis in this study might present the endemic genetic characterization of population structure of the two parasites.

Prevalence of Cryptosporidium baileyi in ostriches (Struthio camelus) in Zhengzhou, China

Few data are available on the molecular characterization of Cryptosporidium spp. in ostriches. The objective of this study was to determine the prevalence of Cryptosporidium species or genotypes in ostriches. A total of 452 fecal samples from five farms, a zoo, and an animal rescue center in Zhengzhou, Henan Province, China were examined for Cryptosporidium oocysts by microscopy of wet mount of fecal materials concentrated by the Sheather's sugar flotation technique. Fifty-three samples were Cryptosporidium-positive from four farms, with an overall prevalence of 11.7%. The percentage of animals shedding oocysts was 0, 16.2%, 7.2%, and 0 in 1–3 weeks, 4–8 weeks, 3–12 months, and more than 12 months ostriches, respectively (χ2=17.74; ρ<0.01). PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene of the 53 Cryptosporidium-positive samples showed the presence of only Cryptosporidium baileyi, which was confirmed by DNA sequencing of the SSU rRNA PCR products from 16 positive samples. Cross-transmission studies demonstrated that the C. baileyi isolate could infect chickens and quails. Thus, ostriches are commonly infected with C. baileyi that is genetically and biologically similar to C. baileyi found in other birds.

Molecular characterization of the Cryptosporidium cervine genotype from a sika deer (Cervus nippon Temminck) in Zhengzhou, China and literature review

A total of 124 fecal specimens were collected from four deer farms in Zhengzhou City, China and examined for Cryptosporidium by Sheather's sugar flotation technique. Cryptosporidim oocysts were detected in two 1-year-old sika deer, and one of the two specimens was genotyped by sequence and phylogenetic analyses of the small subunit ribosomal RNA (rRNA) (18S rRNA), 70-kDa heat shock protein (HSP70), actin, and Cryptosporidium oocyst wall protein (COWP) genes. Results obtained suggested that the Cryptosporidium studied belonged to Cryptosporidium cervine genotype, although slight sequence differences were noticed at the three loci. The similarities between this isolate and other Cryptosporidium cervine genotype isolates were 99.1-99.8%, 9.8%, 99.7%, and 100% at the 18S rRNA, HSP70, actin, and COWP loci, respectively. This study is the first report of Cryptosporidium infection in sika deer in China.

Molecular characterization of a new genotype of Cryptosporidium from American minks ( Mustela vison) in China

A total of 469 fecal samples were collected from American minks ( Mustela vison) on a farm in Hebei Province in China and examined for Cryptosporidium by Sheather's sugar flotation technique and 8 Cryptosporidim isolates were obtained. The partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes of six isolates were sequenced. Sequence data were analyzed together with known Cryptosporidium spp. and genotypes. Results of this multi-locus genetic characterization indicated that the six Cryptosporidium isolates in this study shared the same sequences of the genes studied and were different from known Cryptosporidium species and genotypes. The closest relative was Cryptosporidium ferret genotype with 7, 22, 2 and 2 nucleotide differences in the 18S rRNA, HSP70, COWP and actin genes, respectively. The homology to ferret genotype at the 18S rRNA locus was 99.1%, which is comparable to that between C. parvum and C. hominis (99.2%), or between C. muris and C. andersoni (99.4%). Therefore, the Cryptosporidium in minks in this study is considered a new genotype, the Cryptosporidium mink genotype.

Coccidiosis in goats in the Czech Republic

An observational study was conducted to determine coccidial infections in goats of 13 farms in the Czech Republic. The prevalence of oocysts of Eimeria species in kids (less than 3 months old), weaned but not served goats (from 3 months to 1 year), and adult goats (1 year or more) was determined. Nine Eimeria species were identified in fecal samples by Sheather's sugar flotation technique. The overall prevalence of Eimeria oocysts in fecal specimens was 92.2%. Eimeria arloingi was the most common species with an overall prevalence of 84%, followed by E. hirci (63%) and E. ninakohlyakimovae (56%). Other species present were E. christenseni (55%), E. alijevi (36%), E. caprina (25%), E. aspheronica (12%), E. capriovina (6%) and E. jolchijevi (2%). Two or more Eimeria species were detected in 88% of the samples. The most prevalent species in kids was E. arloingi, while in weaned but not served and adult goats E. ninakohlyakimovae was the most frequently found. The number of oocysts excreted was generally lower in adult goats (2567.3±12 678 OPG), whereas higher number oocyst per gram of feces (OPG) were found in kids (18 565±24 888 OPG). Clinical coccidiosis was detected in two farms, and E. arloingi and E. ninakohlyakimovae were implicated as its cause. Disease was observed in kids 2 to 4 weeks after weaning and watery feces with clumps of mucus, and color changes from brown to yellow or dark tarry, weight loss, and dehydration were the most conspicuous clinical signs. At necropsy, macroscopic changes included mucosal hemorrhages and whitish nodular polyps in the jejunum were found. Histopathological changes were characterized by local hypertrophy and hyperplasia of intestinal villi, villus blunting and inflammatory infiltration in the lamina propria. Numerous developmental stages of the parasites were observed in enterocytes and lacteals of intestinal villi.

Comparison of Staining and Concentration Techniques for Detection of Cryptosporidium Oocysts in Cattle Faecal Specimens

Modified Ziehl Neelsen (MZN), Safranin-Methylene Blue (S-MB), Methylene Blue-Eosin (MB-E), Nigrosin, Carbol Fuchsin and Indirect Fluorescent Antibody (IFA) techniques were compared for detection of Cryptosporidium spp. oocysts in cattle faecal specimens. Of the six techniques, S-MB and IFA techniques were found to be the most efficient. Oocysts stained with S-MB were easily recognized as vivid orange round bodies. The IFA technique offered the advantage of a very readily identifiable apple green fluorescence which provided a definite diagnosis. Comparison of Sheather's sugar flotation, Zinc sulphate (ZnSO4) flotation and Formalin-Ethyl Acetate (FEA) sedimentation techniques revealed that Sheather's sugar flotation technique was the most efficient of the three for concentration of Cryptosporidium spp. oocysts from cattle faecal specimens. Of the negative faecal samples, 12% were found to be positive by Sheather's sugar flotation technique followed by S-MB stain whereas only 7% by ZnSO4 flotation and 6% by FEA sedimentation.

Evidence of an epidemic of Blastocystis hominis infections in preschool children in northern Jordan

Blastocystis hominis is now gaining acceptance as an agent of human intestinal disease. A case-control study of the cause of gastroenteritis in children less than 6 years old was conducted. A total of 500 stool specimens were examined by wet mount preparation, formalin-ether concentration, Sheather's sugar flotation technique, and permanent stains when necessary. B. hominis was found in 63 (25%) of 250 stool specimens of the cases examined; 38 (15%) of these specimens contained this parasite alone. The appearance of severe symptoms was associated with increased numbers of the parasite in the diarrheic specimens (more than five parasites per field at a magnification of x 400). The most common symptoms were abdominal pain, recurrent diarrhea, cramps, anorexia, and fatigue. Contaminated water was suspected to be the major source of infection, since several cases were associated with Giardia infection. These findings support the concept of B. hominis pathogenicity in children with gastroenteritis.