Cryptosporidium spp. infections in livestock and wild animals in Azerbaijan territory.

Cryptosporidium is an intracellular protozoan parasite, globally distributed and capable of infecting various vertebrate species, including humans as well as domestic and wild animals. Cryptosporidium is increasingly gaining attention as a human and an animal pathogen mainly due to its dominant involvement in worldwide waterborne outbreaks. The present paper reviews the current knowledge and understanding of Cryptosporidium spp. in terrestrial and water animals in Azerbaijan.

Cryptosporidium in fish: Implications for aquaculture and beyond.

Aquaculture industries are expanding worldwide and control of Cryptosporidium is of great importance. Cryptosporidiosis is a serious waterborne/foodborne disease, responsible for infectious outbreaks globally. Current knowledge on the Cryptosporidium species in the aquatic environment and their occurrence in piscine hosts is steadily increasing since the Cryptosporidium species have been detected in marine, freshwater, cultured, captive and ornamental fish in a wide range of geographical regions. The zoonotic potential of these parasites and their pathological impact on piscine hosts have been increasingly reported and the fishborne zoonotic risk from Cryptosporidium spp. is of major importance from a public health point of view. Zoonotic subtypes in fish have been described in various studies and are probably related to water contamination from animal and human wastes. This review critically evaluated existing scientific data, related to Cryptosporidium species in piscine hosts, emphasizing transmission routes and the potential impact of piscine cryptosporidiosis in aquaculture. This knowledge will facilitate consumers, authorities and water industries such as fisheries and aquaculture, the prevention and control of waterborne and fishborne cryptosporidiosis in fish products.

Report of Giardia assemblages and giardiasis in residents of Guilan province-Iran.

Giardia duodenalis is considered a highly diverse organism that infects a variety of mammalian hosts. Giardiasis is a significant public health problem in Iran. The purpose of this study was to investigate the occurrence of Giardia duodenalis (G. lamblia, G. intestinalis) infections in humans residing in the Guilan province of Iran. Stool samples were collected during 12 months from 8356 individuals that had been referred to certain hospitals in the capital city of Rasht in the Guilan province, of which 4126 were males and 4230 were females. The samples were separated into three groups according to patient age: group A 1-9 years old (n = 483); group B 10-19 years old (n = 491); and group C greater than 20 years old (n = 7382). The wet mount technique was performed directly on 8356 fecal samples for microscopy. Samples were examined using a saline and iodine direct smear technique in order to confirm the presence of G. duodenalis. The results indicated that 2.5% (206/8356) of the samples were identified as positive for G. duodenalis. A total of 30% of the infected patients (n = 62) had no symptoms. In symptomatic cases, the most common symptoms (46%, n = 95) were abdominal cramps and bloating. Twenty-four percent of patients (n = 50) had cramps, bloating, nausea, and diarrhea. Sixty positive samples were sent for G. duodenalis genotyping based on the amplification of the gdh gene. Forty-one PCR products were successfully selected and sequenced, where 38 (92.6%) samples were identified as genotype A/subgenotype II and in three samples (7.4%) genotype B/subgenotype IV. Genotype A-II had a dominant prevalence as compared to the genotype B-IV samples that were identified in the study. Based on the samples provided by the regional teaching hospitals and subsequent sample analysis, the authors concluded that assemblage A-II is most likely the most common Giardia subgroup infection in the Guilan region. Assemblages have been reported in both humans and animals; however, further studies need to investigate the role of domestic animals and water reservoirs as potential sources of Giardia infection in the Guilan region.

Cryptosporidium genotypes and subtypes distribution in river water in Iran.

Little is known about the diversity and public health significance of Cryptosporidium species in river waters in Iran. In the present study, we determined the genotype and subtype distribution of Cryptosporidium spp. in river water samples in Iran. A total of 49 surface water samples were collected from rivers and surface water in Guilan and Tehran provinces during 2009-2010. Water samples were filtrated through a 1.2-µm pore size membrane filter or by Filta-Max filter followed by immunomagnetic separation or sucrose purification methods. Genotype and subtype of Cryptosporidium were identified by sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively. A total of 24 (48.97%) water samples were positive for Cryptosporidium species by the 18sRNA-based polymerase chain reaction (PCR)-sequencing technique. DNA sequencing revealed the presence of five species of Cryptosporidium (C. parvum, C. hominis, C. muris, C. andersoni, and C. canis) in the water samples of the study area and, to our knowledge, the first report of C. muris in Iran. The results of GP60 gene analysis showed that all C. parvum and C. hominis isolates belonged to the IId and Id subtype families, respectively. The investigated river water supplies were heavily contaminated by pathogenic species of Cryptosporidium from humans and livestock. There is potential risk of waterborne cryptosporidiosis in humans and animals.

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes.

We developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany.

Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2-5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400-6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4',6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0-56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

Evolution of Cryptosporidium in vitro culture.

This overview discusses findings from culturing Cryptosporidium spp. in cell and axenic cultures as well as factors limiting the development of this parasite in cultivation systems during recent years. A systematic review is undertaken of findings regarding the life cycle of the parasite, taking into account physiological, biochemical and genetic aspects, in the hope that this attempt will facilitate future approaches to research and developments in the understanding of Cryptosporidium biology.

Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey.

A total of 70 water samples, including tap, river, fountain and well water were collected in the Ordu province, Middle Black Sea, Turkey and investigated for the detection of Cryptosporidium oocysts. The samples were directly screened microscopically for Cryptosporidium oocysts' detection by immunofluorescence test and subsequently DNA was extracted for the molecular detection by loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Eighteen out of the 70 (25·7%) water samples were found positive for Cryptosporidium spp. by immunofluorescence test and 19 (27·1%) were found positive by LAMP. Nested PCR products were not generated in any of the investigated water samples. A total of 16 randomly selected pellets were spiked with 10 Cryptosporidium oocysts to test the efficiency of the applied method. All the samples were found positive by LAMP for the presence of Cryptosporidium DNA, while the nested PCR assay was positive in only seven (43·75%) out of the 16 examined spiked samples. This is the first report on the occurrence of Cryptosporidium species in environmental and drinking water supplies in the Black Sea area.

Combination of ARAD microfibre filtration and LAMP methodology for simple, rapid and cost-effective detection of human pathogenic Giardia duodenalis and Cryptosporidium spp. in drinking water.

AIMS: In this study, we report a new, simple methodology for the monitoring of Cryptosporidium oocysts and Giardia cysts in drinking water samples, ranging from 10- to 1000-l, which combines a new ARAD microfibre filtration of the (oo)cysts from drinking water and loop-mediated isothermal amplification (LAMP) of a human pathogenic Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium meleagridis and Giardia duodenalis Assemblage A and B specific DNA sequence. METHODS AND RESULTS: During the evaluation of the new concentration and detection technique, spiked reagent and matrix water samples plus blank samples were filtered and tested. In total, 27 samples have been investigated. The results clearly demonstrate that the methodology of using a new ARAD filter, which passed through 1000 l of drinking water with high turbidity (2 NTU), and followed by the LAMP assay was able to detect at least one (oo)cyst in 10 l of drinking water based on a 1000-l sample, taken over a 24-h period. CONCLUSIONS: The described protozoa detection methodology is sensitive, rapid and cost-effective. SIGNIFICANCE AND IMPACT OF THE STUDY: This effective procedure will be useful for small waterworks to achieve continuous monitoring and is also of value for screening catchments to identify those that require further treatment and more detailed microscopic counts.

Detection of Cryptosporidium oocysts in green mussels (Perna viridis) from shell-fish markets of Thailand.

Mussels filter large volumes of water and can concentrate pathogenic organisms, which may act as potential vehicles of transmission to the consumer. A survey study was carried out to investigate the presence of Cryptosporidium protozoan parasites in green mussels (Perna viridis), the smussles pecies most destined for consumption in Thailand. In total, 56 samples were examined from Bangkok (n = 24) and Samut Prakan (n = 32) a wholesale shell-fish markets located at the mouth of the Chao Phraya River. The market for green mussels was closed to the mussel culture placed along the coastal line and this localization may have significant economical impact if the mussels' cultures are found contaminated. Cryptosporidium spp. oocysts were detected by the immunofluorescence antibody method (IFA) in 12.5% of the samples examined. The detection of Cryptosporidium oocysts in green mussels' population of Samut Prakan was higher (15.6%) than in Bangkok market (8.3%). These differences in positive samples from the two locations may be caused by physical, ecological and anthropogenic conditions. This could relay to different contamination levels of marine water by Cryptosporidium oocysts and consequently to contamination of harvested shellfish populations. The results demonstrate that the Cryptosporidium spp. oocysts were found indigenous in mussels from the coastal line of Thailand, indicating that mussels may act as a reservoir of Cryptosporidium foodborne infections for humans.

Rapid identification of Giardia duodenalis by loop-mediated isothermal amplification (LAMP) from faecal and environmental samples and comparative findings by PCR and real-time PCR methods.

The detection of two human-pathogenic Giardia duodenalis assemblages A and B in faecal and water samples by loop-mediated isothermal amplification (LAMP) has been evaluated. The LAMP reaction is reproducible, rapid and specific for the detection G. duodenalis and has lower costs compared to the other molecular assays. This is the first application of LAMP for Giardia detection.

Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR.

The purpose of this study was to estimate the prevalence of equine piroplasmosis in Sudan. The presence of antibodies against Babesia caballi and Theileria equi was determined in serum samples obtained from 158 horses raised in different locations in Sudan by enzyme-linked immunosorbent assay (ELISA). The B. caballi 48-kDa and the T. equi EMA-2 purified recombinant proteins were used as antigens in the ELISA test. Results showed that seven (4.4%) were positive for B. caballi and 80 (63.5%) were positive for T. equi. Polymerase chain reaction (PCR) assays have been applied using primers targeting the B. caballi 48-kDa merozoite antigen, the T. equi SSUrRNA and the T. equi EMA-1 genes. PCR performed on 131 blood spots in filter paper revealed that 33 (25.2%) samples were positive for T. equi but no positives were found for B. caballi. It is concluded that equine piroplasmosis is endemic in the country. This is the first study on serological and molecular epidemiological diagnosis on equine piroplasmosis in Sudan.

First report of Cryptosporidium deer-like genotype in Malaysian cattle.

Fifty faecal samples from diarrheic calves between 1 and 6 months old were collected per rectum from 5 farms around Petaling District in Selangor, Malaysia for Cryptosporidium species detection and genotyping investigation. Oocysts were purified using sedimentation and gradient centrifugation, then examined by immunofluorescence assay (IFAT). Genomic DNA was extracted from all samples and nested PCR was performed to amplify the SSU rRNA gene. Eighteen samples (36%) were positive for Cryptosporidium species by PCR. The sequence and phylogenetic analysis of 14 isolates indicated that Cryptosporidium parvum was most common (11 isolates) followed by Cryptosporidium deer-like genotype (3 isolates). The present work reports the first data on Cryptosporidium genotyping from cattle in Malaysia.

First investigations into the prevalence of Cryptosporidium and Giardia spp. in Hungarian drinking water.

Safe drinking water is a top priority in preventing disease outbreaks and is of general concern to everyone. This study examines the occurrence of Cryptosporidium and Giardia in Hungarian drinking water supplies for the first time. A total of 76 raw and drinking water samples were examined using the U.S. EPA Method 1623. From these 15 of 34 (48.4%) raw water samples tested positive for Giardia and 7 (26.6%) for Cryptosporidium. Twelve of 45 (26.7%) drinking water samples were positive for Giardia and 6 (13.3%) for Cryptosporidium. Overall, Giardia cysts and/or Cryptosporidium oocysts were detected in 48% of the raw water samples and 35% of the drinking water samples. The highest levels in drinking water were found to be 3 oocysts/100 litres of Cryptosporidium and 63.6 cysts/100 litres for Giardia, enough to cause giardiasis. The highest levels in raw water were 1,030 cysts/100 litres for Giardia and 50 oocysts/100 litres for Cryptosporidium and higher oocyst densities were associated with source water receiving effluents from sewage treatment plants or originating from a forest environment. In addition to this monitoring, riverbank filtrated water and raw water from the River Danube in Budapest were monitored in order to ascertain protozoan removal efficiency of riverbank filtration (RBF). A total of 157 samples, including 87 samples from the River Danube and 70 samples post RBF, were examined. Cryptosporidium and Giardia were detected regularly in the river water but never in riverbank filtered water suggesting the effectiveness of RBF as a purification method. The occurrence of Cryptosporidium oocysts and Giardia cysts in the investigated water supplies may require the water utilities and water authorities in Hungary to apply additional monitoring and treatment and/or watershed controls.

Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material.

It is possible to detect and distinguish Leishmania parasites using PCR-RLFP - a combination of PCR and analysis of the fragment-length polymorphism seen when the amplicons are digested with one or more restriction enzymes. In the present study, clinical samples from 24 Jordanians suspected to have cutaneous leishmaniasis and cultures set up using leishmanial parasites from five Greek dogs were investigated using PCR, in which the internal-transcribed-spacer-1 (ITS1) region of the parasites' ribosomal-RNA gene was amplified, followed by HaeIII digestion of the resulting amplicons. The cultures, which were all maintained in Leibowitz L-15 medium with 20% foetal calf serum, were each investigated as serial dilutions. Using the PCR-RLFP analysis, each culture was identified as L. donovani and each was found positive for this parasite with a mean sensitivity of 66%-100% (depending on the culture dilution tested), a specificity of 100%, a mean positive predictive value of 100%, and a negative predictive value of 74.6%-100%. When simulated clinical samples, created by mixing human blood with known numbers of L. donovani promastigotes, were investigated, the PCR-RFLP gave optimal results (with a value of 100% each for sensitivity, specificity and positive and negative predictive values). When the real clinical samples (25 lesion aspirates and 20 samples of peripheral blood from 24 Jordanian patients) were investigated using the molecular method, 20 (84%) of the patients were found to have lesion aspirates that were PCR-RFLP-positive for L. major (although, by microscopy, only six were found to have amastigote-positive lesion aspirates). None of the blood samples from the Jordanian patients, however, was found PCR-positive.

Evaluation and applicability of a purification method coupled with nested PCR for the detection of Toxoplasma oocysts in water.

AIMS: To describe the development, evaluation and applicability of a complete method for the detection of Toxoplasma gondii in water. METHODS AND RESULTS: The method incorporated concentration of water samples by Al(2)(SO(4))(3)-flocculation, purification by discontinuous sucrose gradients and detection of toxoplasmic DNA by 18S-rRNA nested PCR. Tap water replicates and natural water samples were seeded with defined numbers of Toxoplasma oocysts and processed for evaluation studies. When applied to environmental samples, the method gave highest detection sensitivities of 100 oocysts in river water and 10 oocysts in well- and sea water. The method was finally applied in 60 water samples of different quality and origin collected over a 14-month period. Toxoplasmic DNA was detected in four samples. CONCLUSIONS: The method offers an alternative towards improving current methods that can be used for the detection of Toxoplasma oocysts in environmental water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method in its current form will be helpful for assessment of Toxoplasma contamination in water resources, particularly after outbreak events.

Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria.

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.

Seasonal variation in Sarcocystis species infections in goats in northern Iraq.

We investigated the prevalence of sarcocystosis in 826 goats slaughtered in the winter season from November to April in northern Iraq. The prevalence of macrocysts was on average 34%, with only 20% infected animals in November, but 46% in February. The infection rate in 1-, 3- and 6-year-old goats was 4%, 48%, and 83%, respectively. The highest specificity of infection was in the oesophagus (99%) and the lowest in the diaphragm (3%). Grossly, we identified 2 forms of macroscopic sarcocysts, fat and thin, with different morphological characteristics. The prevalence of microcysts was 97% and no effects of age, sex and seasonal variations were observed. Development of microcysts in the small intestine of dogs and cats has also been investigated. The pre-patent period in experimentally infected dogs was 12-14 days and the patent period lasted 64-66 days. A dog shed about 155 million sporocysts, but no sporocysts were shed by cats that had been fed the same infected tissues, thus identifying the microcysts as Sarcocystis capracanis.

Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water.

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1 -L water replicates and concentrated using centrifugation, Al2(SO4)3 or Fe2(SO4)3 flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomal RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al2(SO4)3 flocculation. No PCR products were generated for samples that had been pre-treated using Fe2(SO4)3 flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.