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Background: Hypertension is an important public health challenge worldwide. It is the most common cardiovascular disease, and the most important modifiable risk factor for cardiovascular, cerebrovascular and renal diseases. Objective: The aim of the study was to determine the prevalence of Hypertension among adult patients attending the General Outpatient Clinics of the University of Uyo Teaching Hospital Uyo, South-South Nigeria Materials and Methods: A cross-sectional study of three hundred and eighty-five (385) adults (18 years and above) attending the General Outpatient Clinic of the University of Uyo Teaching Hospital, Uyo, Nigeria, was carried out between March and June, 2013. The study subjects were administered Semi- structured questionnaires which sought information on socio-demographic characteristics and the presence or absence of hypertension. Data analysis was done using the Statistical Package for Social Sciences (SPSS) version 20.0. Results: The study subjects consisted of 166 males and 219 females (male: female = 1:1.3). The mean age of respondents was 37.7 years with a standard deviation of 14.4. The overall prevalence of hypertension was 30.1%. Conclusion: The prevalence of hypertension is high. There is need for continuous public enlightenment on preventive measures by health care givers to reduce the prevalence of this silent but deadly disease.

Background: Under five morbidity constitutes serious public health challenge with attendant deleterious sequelae on children globally, especially in sub-Saharan Africa and other least developed countries. This is further aggravated by inadequate research data on the state of health of under five children especially in rural areas. This study was done to describe the pattern of morbidity among under five children presenting at a rural secondary health care facility in Etinan Local Government Area of Akwa Ibom State, Southern Nigeria. Method: This was a descriptive retrospective study involving 494 children aged 0-59 months who presented at the general out patient and emergency units of Qua Iboe Church Leprosy Hospital (QICLH) Ekpene Obom, Etinan Local Government Area, Akwa Ibom State, between June 2014 to June, 2017. The case records of the under five children who presented at the period of the study both at the out patient and the wards were collated and entered into a spread sheet. The age, sex, diagnoses, causes and outcome of admission were extracted from the records. Results: The mean age of the children was 21.63 ( ±15.34) months. Results obtained showed that 259 (52.43%) of the children were male while 235 (47.57%) were female, giving a male to female ratio of 1.1:1. Out of 526 morbidities presented, 262 (49.81%) were malaria, 133 (25.29%) were acute respiratory infections (ARI’s), 37(7.03%) were skin infections, 23(4.37%) were diarrhoea while haemoglobinopathy was the least presentation of only one case (0.19%). There were 74 cases of admission, giving an admission rate of 14.17%. Complicated malaria was the commonest cause of admission while 89.19% of those admitted were successfully treated and discharged. Conclusion: Most of the morbidities that presented in the facility during the period of the study were preventable through improved socioeconomic condition, health education and immunization. The need therefore to improve the socioeconomic conditions of the populace especially those aspects that affect the wellbeing of under five children and strict adherence to immunization programme as a panacea for reduction of morbidity among the under five children in rural areas is recommended.

In this article, the atom excitation suppression is studied in two mechanisms. The first mechanism for excitation suppression is caused by an external DC electric field. The second mechanism is due to the energy shift caused by an electric field generated by free charges, which are created by ionizing atoms. The latter mechanism is known as the Coulomb blockade. Here, the Coulomb forces originate from ions created by ionizing atoms with a UV laser. The interaction, which causes the suppression, is treated theoretically as dipole–charge interactions. In the model, the charge is an ion, and the dipole is an atom. From measurements, we use 85Rb atoms. The valence electron and the ion core are the two poles of an electric dipole. The interaction potential energy between the ion and the atom is proportional to 1R2, and the frequency shift caused by this interaction is proportional to 1R4, where R is the distance between the ion and the dipole considered. This research is motivated by potential applications for quantum information storage, remote control, creating hot plasmas using cold atoms, as well as electronic devices.

Background: Most neonatal deaths occur in low and middle-income countries (LMICs). These deaths can be prevented through universal access to basic high-quality in-patient health services. Prematurity, neonatal sepsis, and perinatal asphyxia have been reported as the leading causes of in-patient neonatal deaths. This study aimed to assess the trend of neonatal mortality in our hospital, determine the pattern and causes of neonatal mortality, and evaluate the factors associated with neonatal mortality in our facility., Methodology: This was a retrospective cross-sectional descriptive study conducted in the Special Care Babies Unit (SCBU) and Sick Babies Unit (SBU) of the University of Uyo Teaching Hospital, over seven years (2015-2021). Demographic, clinical, and mortality data was extracted from the case record files of patients into a structured proforma and analysed., Results: There was a total of 228 deaths comprising 130 males (57.02%) and 98 (42.98%) females. The median age at demise was 4.00 (IQR = 1.00 - 12.00) days for both genders. The majority (71.50%) of deaths occurred in the Sick Babies Unit. More males died than females (57% vs 43%). The three leading causes of death were: prematurity (38.60%), neonatal sepsis (38.16%), and birth asphyxia (13.60%)., Conclusion: The leading causes of neonatal mortality in our environment are prematurity and neonatal sepsis. There is a need for increased community education on antenatal care, training of traditional birth attendants, improved newborn transportation facilities, and provision of neonatal intensive care facilities., (Copyright © 2024 Nigerian Medical Association.)

Background: Hypertension is a modifiable risk factor for cardiovascular, cerebrovascular and renal diseases., Objective: The objective of the study was to determine the risk factors for Hypertension among adults attending the general outpatient clinic of University of Uyo Teaching Hospital., Methods: A cross-sectional study of three hundred and eightyfive (385) adults (18 years and above) attending the General Outpatient Clinic of the University of Uyo Teaching Hospital, Uyo, Nigeria, was carried out between March and June, 2013. Information on socio-demographic characteristics, presence or absence of hypertension, symptom counts, duration of illness as well as risk factors for hypertension were sought., Results: The study 385 subjects consisted of 166 males and 219 females (male: female = 1:1.3). The mean age of respondents was 37.7± 14.4 years. After multivariate analysis, age, family history of hypertension and obesity were identified as independent risk factors for hypertension in this study., Conclusion: There is need for public enlightenment on the essence of routine screening of family members of hypertensive patients, avoidance of obesity and reduction of salt intake in our meals.

Background: In the last few decades, the evaluation of the quality of life (QoL) among older adults has become increasingly important in health as well as in social sciences. There has been growing emphasis on the need to understand what influences older people's QoL as it is argued to be of greater value than the traditional outcome measures, such as health status., Aim and Objective: The aim of this study was to determine the sociodemographic factors affecting the QoL of elderly patients attending the General Outpatient Clinics of the University of Uyo Teaching Hospital (UUTH), Uyo., Methodology: The study was a cross-sectional descriptive study. Three hundred and ten elderly persons attending the General Outpatient Clinics of the UUTH for medical conditions between July and September 2014 were consecutively recruited for the study. Details of sociodemographic information were taken, and QoL of respondents was assessed using the older persons' QoL questionnaire. Data were analyzed using the Statistical Package for Social Sciences-17.0, and the level of statistical significance was set at P < 0.05., Results: Of the three hundred and ten respondents recruited for the study, One hundred and seventy-seven (57.1%) were female and one hundred and thirty-three (42.9%) were male. The female-to-male ratio was 1.3:1. The age range of the respondents was between 60 and 90 years and the mean age (±standard deviation) was 67.4 (±6.6) years. Two hundred and thirty-two respondents (74.8%) had at least primary level of education and one hundred and sixty-one (51.9%) were married. The median monthly income was ₦ 25,500.00 with interquartile range of ₦ 10,000.00 to ₦ 50,000.00. From the study, 85.5% of the elderly reported an overall good QoL. High-income grade ( P = 0.019), high social class ( P = 0.036), and high level of education ( P < 0.001) were the factors associated with good QoL in this study on univariate analysis., Conclusion: A high percentage of respondents reported an overall good QoL. High-income grade, high social class, and high level of education were the factors associated with good QoL., Competing Interests: There are no conflicts of interest.

The environmental distribution of Giardia spp. and Cryptosporidium spp. is dependent upon human, agricultural, and wildlife sources. The significance of each source with regard to the presence of parasites in the environment is unknown. This 2-year study examined parasite prevalence in human sewage influent, wildlife, and agricultural sources associated with the North Saskatchewan River Basin in Alberta, Canada. Fecal samples were collected from cow-calf, dairy, and hog operations in the watershed area. Sewage-treatment facilities were sampled bimonthly during the 2-year study, and wildlife scat was collected at locations along tributaries of the North Saskatchewan River. All samples were analyzed for the presence of Giardia and Cryptosporidium, using sucrose-gradient separation followed by immunofluorescent microscopy. Giardia and Cryptosporidium were detected in all three sources. The lowest prevalence of both Giardia (3.28%) and Cryptosporidium (0.94%) was found in wildlife, with 6 of 19 species testing positive. Sewage influent had the highest prevalence of Giardia (48.80%) and Cryptosporidium parvum-like oocysts (5.42%); however, the concentration of both parasites was minimal compared with the concentration detected in cattle feces. Cow-calf sources contained the highest concentration of Giardia (mean 5800/g feces, P < 0.01), and dairy sources contained the highest concentration of C. parvum-like oocysts (mean 295/g feces, P < 0.01). Although prevalence and concentration are higher in cattle feces than in sewage, the Giardia and Cryptosporidium in animal manure do not have direct access to water draining into the North Saskatchewan River. PCR-based characterization of rDNA from isolates of Cryptosporidium collected from Alberta human, pig, calf, mature steer, dog, cat, and beaver hosts revealed distinct genetic differences that may reflect host specificity.

Confusion exists over the species status and host-specificity of the tapeworm Rodentolepis (= Hymenolepis) nana. It has been described as one species, R. nana, found in both humans and rodents. Others have identified a subspecies; R. nana var. fraterna, describing it as morphologically identical to the human form but only found in rodents. The species present in Australian communities has never been identified with certainty. Fifty one human isolates of Rodentolepis (= Hymenolepis) nana were orally inoculated into Swiss Q, BALB/c, A/J, CBA/ CAH and nude (hypothymic) BALB/c mice, Fischer 344 and Wistar rats and specific pathogen free (SPF) hamsters. Twenty four human isolates of R. nana were cross-tested in flour beetles, Tribolium confusum. No adult worms were obtained from mice, rats or hamsters, even when immunosuppressed with cortisone acetate. Only one of the 24 samples developed to the cysticercoid stage in T. confusum; however, when inoculated into laboratory mice the cysticercoids failed to develop into adult worms. The large sample size used in this study, and the range of techniques employed for extraction and preparation of eggs provide a comprehensive test of the hypothesis that the human strain of R. nana is essentially non-infective to rodents.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.

The aim of this prospective study was to determine the prevalence of Cryptosporidium parvum in hospitalized children in Kota Bharu, Malaysia. Over a 19 month study period, 258 stool samples were examined from 159 children; 109 with diarrhea and 50 controls without diarrhea. Modified Ziehl-Neelsen staining method and a polymerase chain reaction (PCR) assay were used to detect C. parvum and the samples were also examined for the presence of other intestinal parasites. Only 1 of the 109 (0.9%) children with acute diarrhea was positive for C. parvum by microscopy and PCR. Thirty-one percent of children were infested with other intestinal parasites, the most common being Ascaris lumbricoides and Trichuris trichiura. In conclusion, we found C. parvum to be an uncommon infective agent in hospitalized children with or without diarrhea in Kota Bharu, Malaysia.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.

Nucleotide sequences of the Cryptosporidium oocyst wall protein (COWP) gene were obtained from various Cryptosporidium spp. (C. wrairi, C. felis, C. meleagridis, C. baileyi, C. andersoni, C. muris, and C. serpentis) and C. parvum genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of Cryptosporidium spp. and genotypes.

Understanding the epidemiology of zoonotic parasitic infections is dependent upon the availability of accurate and sensitive diagnostic techniques. The development of molecular diagnostic methods, particularly those utilising PCR for the detection of zoonoses will contribute greatly to the identification and control of these pathogens, by increasing the speed of diagnosis, specificity and sensitivity, reproducibility and ease of interpretation. Molecular characterisation studies allow us to distinguish between closely related infectious agents and to document the patterns of transmission of 'strains' and species within populations. This will allow precise determinations to be made about the aetiological agent, its characteristics and the source of infection. This review focuses on recent detection and characterisation techniques for both emerging and re-emerging parasite zoonoses.

There is controversy in the taxonomy of Cryptosporidium parasites and the public health significance of Cryptosporidium isolates from various animals. Recent advances in molecular characterization of Cryptosporidium parasites have allowed the re-examination of species structure of the genus Cryptosporidium. Non-parvum Cryptosporidium spp and new C. parvum genotypes in immunocompromised humans can now be clearly detected. In this article, Lihua Xiao and colleagues summarize the current biological and molecular evidence for different Cryptosporidium spp, and the public health importance of these species and new C. parvum genotypes.

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.

Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

Genetic and phylogenetic characterization of Cryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the "dog" genotype is, in fact, a valid species.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Objective: To perform a morphological and genetic characterisation of a Cryptosporidium infection in an Indian ring-necked parrot (Psittacula krameri) and to compare this with C meleagridis from a turkey., Design: Tissue and intestinal sections from an Indian ring-necked parrot were examined microscopically for Cryptosporidium. The organism was also purified from the crop and intestine, the DNA extracted and a portion of the 18S rDNA gene amplified, sequenced and compared with sequence and biological information obtained for C meleagridis from a turkey as well as sequence information for other species of Cryptosporidium., Results: Morphological examination of tissue sections from an Indian ring-necked parrot revealed large numbers of Cryptosporidium oocysts attached to the apical border of enterocytes lining the intestinal tract. Purified Cryptosporidium oocysts measured about 5.1 x 4.5 microns, which conformed morphologically to C meleagridis. The sequence obtained from this isolate was identical to sequence information obtained from a C meleagridis isolate from a turkey., Conclusion: Cryptosporidium meleagridis was detected in an Indian ring-necked parrot using morphological and molecular methods. This is the first time that this species of Cryptosporidium has been reported in a non-galliform host and extends the known host range of C meleagridis.

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.

Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodenalis Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

A 298-bp region of the Cryptosporidium parvum 18S rRNA gene and a 390-bp region of the acetyl coenzyme A synthetase gene were sequenced for a range of Cryptosporidium isolates from wild house mice (Mus domesticus), a bat (Myotus adversus), and cattle from different geographical areas. Previous research has identified a distinct genotype, referred to as the "mouse"-derived Cryptosporidium genotype, common to isolates from Australian mice. Comparison of a wider range of Australian mouse isolates with United Kingdom and Spanish isolates from mice and cattle and also an Australian bat-derived Cryptosporidium isolate revealed that the "mouse" genotype is conserved across geographic areas. Mice are also susceptible to infection with the "cattle" Cryptosporidium genotype, which has important implications for their role as reservoirs of infection for humans and domestic animals.

The Cryptosporidium ITS1, 5.8S and ITS2 rDNA regions from a number of Cryptosporidium isolates from different hosts and geographical areas were cloned and sequenced in order to investigate the extent of sequence heterogeneity between human and cattle-derived isolates from different geographical locations and also between isolates of Cryptosporidium from different hosts such as cats, pigs, mice and a koala. Calf-derived isolates from different continents were virtually identical as were human-derived isolates from the UK and Australia. Genetic differences between Cryptosporidium isolates were extensive and were in fact greater than the level of nucleotide divergence between Toxoplasma gondii and Neospora caninum rDNA sequences. Based on the sequence information derived from this study, PCR-RFLP of the ITS1 region was undertaken in order to directly amplify and genotype Cryptosporidium isolates from different hosts. This PCR-RFLP approach can now be used for molecular epidemiology studies, circumventing the need for costly sequencing and allowing a wider range of genetically different isolates to be examined.

In this article, Una Morgan and Andrew Thompson briefly review the latest information on polymerase chain reaction (PCR)detection of Cryptosporidium parvum in both clinical and environmental samples. Current detection methods for Cryptosporidium are cumbersome, time-consuming and lack sensitivity. A variety of PCR tests have been described recently in the literature and this article discusses the advantages and disadvantages of each new technique and their potential for future diagnosis of Cryptosporidium.

The genetic analysis of oocysts recovered from the stools of humans and animals infected with Cryptosporidium parvum has consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis, C. parvum isolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species C. parvum.

A 298 bp region of the Cryptosporidium parvum 18S rDNA and a 390 bp region of the acetyl-CoA synthetase gene were sequenced for a range of human and animal isolates of Cryptosporidium from different geographical areas. A distinct genotype is common to isolates from cattle, sheep and goats and also an alpaca from Peru and is referred to here as the 'calf'-derived Cryptosporidium genotype. Another genotype of 'human'-derived isolates also appears to be conserved amongst human isolates although humans are also susceptible to infection with the 'calf' Cryptosporidium genotype. Mice and pigs carry genetically distinct genotypes of Cryptosporidium. Three snake isolates were also analysed, 2 of which exhibited C. muris genotypes and the third snake isolate carried a distinct 'mouse' genotype.

Faecal samples were collected from domestic cats in the metropolitan area of the city of Perth, Western Australia, and screened for the presence of Cryptosporidium by both microscopy and PCR. Of 162 samples screened, two were positive for Cryptosporidium (a prevalence of 1.2%). Sample Ct33 was from an 18-month-old female and sample Ct131 from a 12-month-old female. Morphological studies revealed oocysts with an average size of 4.6 x 4.0 microm, smaller in size than isolates typically seen in humans (5.0 x 4.5 microm). Sequence analysis of PCR products showed sequences from cat isolates to be different to previously sequenced human and calf isolates, with cat isolates exhibiting 8.1% sequence divergence from these isolates. Phylogenetic analysis grouped the cat isolates into a distinct group, separate from other C. parvum isolates and Cryptosporidium species. These results lend support to the existence of a cat-adapted Cryptosporidium strain or species.

PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.

The development of molecular diagnostic methods, particularly those utilizing PCR for the detection of parasitic protozoa will contribute greatly to the identification and control of these pathogens, by increasing speed of diagnosis, specificity and sensitivity, reproducibility and case of interpretation. PCR methods are not without their problems however, and there is a need for laboratory procedures to be refined before PCR-based assays are accepted as the tools of choice for the routine detection of protozoan parasites. The application of PCR detection to various parasites is discussed.

In this chapter, the contribution of molecular tools in understanding the aetiology and ecology of infectious diseases is examined in the context of molecular epidemiology (ME). ME is seen as providing the 'tools', both laboratory and analytical, which have predictive significance in epidemiological investigations of the causation of disease. A diversity of questions can be addressed with these tools which can conveniently be viewed as particular regions of DNA and grouped according to the different hierarchical levels of specificity by which infectious agents can be characterized. These groupings and the applications of the different molecular tools are described, and consideration given to the most appropriate methods of analysing data from ME investigations.

Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.

Following the first report of avian Giardia infection in Australia, isolates of the parasite recovered from naturally infected straw-necked ibis (Theskiornis spinicollis) were characterized using median body morphology, scanning electron microscopy, multilocus enzyme electrophoresis, random amplified polymorphic DNA (RAPD), and small subunit ribosomal RNA (SSU-rRNA) analyses. Results were compared with Giardia from other birds and mammals, and the extent of genetic diversity between a range of ibis isolates collected in Western Australia was determined. The ibis isolates of Giardia were genetically relatively homogeneous, which is in contrast to the extensive genetic heterogeneity often displayed by mammalian Giardia isolates. Morphologically, Giardia from ibis were similar to Giardia ardeae although they differed genetically and by the fact that the ibis isolates could not be established in in vitro culture. Sequence data of the DNA coding for the SSU-rRNA found a 96% homology between the ibis isolates from Western Australia and G. ardeae, suggesting that they represent distinct strains of the same species. In contrast, the ibis isolates were genetically and morphologically very different than Giardia duodenalis and Giardia muris from mammals.