Toxoplasma gondii in the faeces of wild felids from the Atlantic Forest, Brazil

BACKGROUND Toxoplasma gondii is a apicomplexan parasite of virtually all warm-blooded species. All true cats (Felidae) can act as definitive hosts for this parasite by shedding resistant oocysts into the environment. However, the patterns of oocysts shedding are only partially understood in domestic cats and largely unknown in wild felids. OBJECTIVES We carried out molecular analysis of 82 faecal samples from wild felids collected in the Serra dos Órgãos National Park (Parnaso), Rio de Janeiro, Brazil. METHODS We screened samples for T. gondii DNA using a quantitative polymerase chain reaction (qPCR) targeting the 529bp DNA fragment. Polymerase chain reaction (PCR)-positive samples were genotyped using 15 microsatellite markers. RESULTS Only one faecal sample from a Puma yagouaroundi was PCR-positive [cycle threshold (Ct) = 26.88]. This sample was contaminated by a T. gondii strain of BrIII lineage, a common lineage in domestic animals from Brazil. MAIN CONCLUSIONS This first report of T. gondii in faeces of wild South American felids in their natural environment indicates infrequent oocyst shedding and suggests a role of acquired immunity in limiting re-excretion as in domestic cats. The presence of a domestic strain of T. gondii in a faecal sample from a wild felid at very low concentrations (not detected by microscopy) is consistent with the hypothesis of host-parasite co-adaptations limiting the circulation of T. gondii strains between domestic and wild environments.

Copro-ovoscopical Assessment of Gastrointestinal Parasitism in Captive Canine and Feline Carnivorans

The health surveillance through a copro-ovoscopical study was conducted to assess the gastrointestinal parasitic infections in captive canine {jackal (Canis aureus), wolf (Canis lupus), dhole (Cuon alpinus) and hyaena (Hyaena hyaena)} and feline carnivorans {lion (Panthera leo), tiger (Panthera tigris), leopard (Panthera pardus) and jungle cat (Felis chaus)}, maintained at Bannerghatta Biological Park, Bengaluru, Karnataka. A total of 41 fecal samples from apparently normal/healthy captive lions, 35 from tigers, 34 from leopards, 14 from jungle cats, 4 from dholes, 4 from wolves, 4 from jackals and 2 from hyenas were collected over a period of 12 months during 2015-2016 and were screened using classical parasitological techniques including sedimentation and floatation technique followed by microscopic identification of eggs. It revealed the prevalence of ova of 3 (7.31%) Strongyle sp., 8 (19.51%) Ancylostoma sp., 21 (51.21%) Toxocara sp., 16 (39.02%) Toxascaris leonina, 4 (9.75%) mixed (Ascaris and Ancylostoma sp.) in lions; 19 (54.28%) Toxocara sp., 8 (22.85%) Toxascaris leonina, 6 (17.14%) Ancylostoma sp., 2 (5.71%) oocyst of Eimeria sp., 2 (5.71%) Spirometra sp. in tigers; 13 (38.23%) Toxocara sp., 6 (17.64%) Ancylostoma sp. in leopards; 7 (50%) Isospora sp. and 2 (14.28%) Toxocara sp. in jungle cats; 2 (50%) Ancylostoma sp. in jackals; 2 (50%) Toxocara canis and 1 (25%) Ancylostoma sp. in wolves; 2 (50%) Toxocara canis in dholes; while ova of only 1 (50%) Ancylostoma sp. could be observed in hyaena. This preliminary data may be useful for health management of the said species in captivity.

Excretion of Toxoplasma gondii oocysts from Feral Cats in Korea

Sporulated oocysts from the feces of infected cats with Toxoplasma gondii can cause detrimental disease in both humans and animals. To investigate the prevalence of feral cats that excrete T. gondii oocysts in the feces, we examined fecal samples of 563 feral cats over a 3-year period from 2009 to 2011. Oocysts of T. gondii excreted into the feces were found from 4 of 128 cats in 2009 (3.1%) and one of 228 (0.4%) in 2010 while none of the 207 cats in 2010 were found positive with oocysts in their feces, resulting in an overall prevalence rate of 0.89% (5/563) between 2009 and 2011. Among the 5 cats that tested positive with T. gondii oocysts, 4 of the cats were male and 1 was a female with an average body weight of 0.87 kg. Numerous tissue cysts of 60 μm in diameter with thin (<0.5 μm) cyst walls were found in the brain of one of the 5 cats on necropsy 2 months after the identification of oocysts in the feces. A PCR amplification of the T. gondii-like oocysts in the feces of the positive cats using the primer pairs Tox-5/Tox-8 and Hham34F/Hham3R confirmed the presence of T. gondii oocysts in the feces. This study provides a good indication of the risk assessment of feral cats in the transmission of T. gondii to humans in Korea.

Eimeria intricata Spiegel, 1925 em ovinos de Mossoró, Rio Grande do Norte, Brasil / Eimeria intricata Spiegel, 1925 in sheep Mossoro, Rio Grande do Norte, Brazil

Objetivou-se identificar através da morfometria, espécimes de Eimeria em ovinos. Realizou-se oocistograma (OOPG) em 50 ovinos da raça Dorper de Mossoró, RN. As amostras fecais positivas no OOPG foram submetidas à esporulação em solução aquosa de bicromato de potássio 2,5% por sete dias, sob temperatura ambiente (≅ 27ºC). Foi feita identificação de 100 oocistos selecionados aleatoriamente no exame de microscopia óptica (objetiva de 40X, fator de correção 0,333). Os dados foram expressos em média, desvio padrão, valores mínimos e máximos, calculados pelo programa estatístico SPSS (Statistical Package for the Social Sciences) 21.0. Os coccídeos, objeto deste trabalho, classificados em Eimeria intricata Spiegel, 1925 apresentaram: oocisto com média de comprimento 50,83µm (43,29-53,28µm); largura média 36,18µm (33,30-39,96µm) e índice morfométrico médio 1,40 (1,18-1,60); esporocisto com média de comprimento 19,09µm (16.65-9,98µm); largura média 11,98 (9,99-13,32µm) e índice morfométrico médio 1,60µm (1,50-2,0µm). Este registro amplia o conhecimento da ocorrência de E intricata em mais uma localidade do Nordeste brasileiro e auxilia a reconhecer que a existência da mesma nos rebanhos ovinos do Rio Grande do Norte pode não desencadear quadros patogênicos, mas indica falhas no manejo dos animais.

The objective of this study was to identify morphometry, Eimeria specimens of Dorper sheep of Mossoró, RN. Oocyst (OOPG) was performed on 50 sheep. Positive faecal samples in the OOPG were submitted to sporulation in aqueous solution of 2.5% potassium dichromate for seven days, at room temperature (≅ 27ºC). Identification of 100 randomly selected oocysts was performed on the optical microscopy (objective 40X, correction factor 0.333). Data were expressed as mean, standard deviation, minimum and maximum values, calculated by the statistical program SPSS (Statistical Package for the Social Sciences) 21.0. The coccidia, object of this work, classified in Eimeria intricata Spiegel, 1925 presented: oocyst with average length 50.83µm (43.29-53.28µm); Mean width 36.18µm (33.30-39.96µm) and mean morphometric index 1.40 (1.18-1.60); Sporocyst with a mean length of 19.09µm (16.65-9.98µm); Mean width 11.98 (9.99-13.32µm) and mean morphometric index 1.60µm (1.50-2.0µm). This record amplifies the knowledge of the occurrence of E.intricata in another locality of the Northeast of Brazil and helps to recognize that the existence of the same in the sheep flocks of Rio Grande do Norte may not trigger pathogenic conditions, but indicates failures in the management of the animals.

Risk factors for Toxoplasma gondii infection in sheep in the northeastern region of Brazil / Fatores de risco para a infecção de Toxoplasma gondii em ovinos da região nordeste do Brasil

Toxoplasma gondii is an infective parasite that causes reproductive disorders such as abortion, fetal mummification, birth of weak offspring, and stillbirth, thereby causing economic losses to sheep production. The northeastern region of Brazil has approximately 171 million small ruminants, of which 5.4% are sheep. The present study aimed at determining the rate of occurrence of T. gondii in sheep flocks on 60 farms in 19 municipalities in the three mesoregions (eastern, semi-arid, and sertão or backlands) of the state of Sergipe, Brazil, and the risk factors associated with this infection. Serum samples were collected between 2011 and 2012, from 60 farms located in 19 municipalities in the three mesoregions: 680 in the eastern region, 280 in the semi-arid region, and 240 in the backlands, totaling 1,200 samples (990 females and 210 males). Anti-T. gondii antibodies were detected by means of the indirect fluorescence antibody test (IFAT ≥ 64). The highest occurrence was detected in the eastern region (45.3%, p = 0.001). On farms with subsistence production, the risk of having animals infected by T. gondii was approximately twice as high as on breeding/rebreeding/fattening farms (OR: 3.03; CI: 1.97-4.68). There was a significant lack of sanitary care, such as absence of a dunghill (p = 0.000; OR: 1.60; CI: 1.26-2.03), quarantine (p = 0.000; OR: 1.87; CI: 1.45-2.41) and disinfection (p = 0.003; OR: 1.46; CI: 1.13-1.88). Regarding feeding, the risk of infection was 1.74 and 1.37 times higher in places that used a trough and/or that cats could access, respectively. The present study allows the conclusions that T. gondii is found on farms in the three mesoregions of the state of Sergipe and that environmental and management factors have an influence on sheep infection.(AU)

Toxoplasma gondii é um parasita cuja infecção leva a desordens reprodutivas como aborto, mumificação fetal, nascimento de cordeiros fracos e natimortos, provocando perdas econômicas na produção ovina. A região nordeste do Brasil possui aproximadamente 171 milhões de pequenos ruminantes, dos quais 5,4% são ovinos. Este estudo tem como objetivo determinar a ocorrência de T. gondii nos rebanhos ovinos de 60 propriedades de 19 municípios de três mesorregiões (leste, semiárido e sertão) do estado de Sergipe, Brasil, e os fatores de risco associados a essa infecção. Amostras de soro foram coletadas entre 2011 e 2012, em 60 propriedades localizadas em 19 municípios das três mesorregiões: 680 na região leste, 280 no semiárido e 240 no sertão, totalizando 1.200 amostras (990 fêmeas e 210 machos). Anticorpos anti-T.gondii foram detectados por reação de imunofluorescência indireta (RIF ≥ 64). A maior ocorrência foi detectada na região leste (45,3%, p = 0.001). Em propriedades com produção de subsistência, o risco de animais infectados por T. gondii é aproximadamente duas vezes maior que nas de cria/recria/engorda (OR = 3.03/ IC: 1.97-4.68). A ausência de cuidados sanitários, como ausência de esterqueira (p = 0.000/ OR: 1.60; CI: 1.26-2.03); quarentena (p = 0.000/ OR: 1.87; CI: 1.45-2.41) e desinfecção (p = 0.003/ OR: 1.46; CI: 1.13-1.88) foram significantes. Em relação à alimentação, o risco de infecção aumenta 1.74 e 1.37 em locais que utilizam cocho ou com presença de gatos, respectivamente. Este estudo permite concluir que o T. gondii é encontrado em propriedades das mesorregiões do estado de Sergipe e fatores ambientais e de manejo estão influenciando nas infecções em ovinos.(AU)

HUMAN TOXOPLASMOSIS OUTBREAKS AND THE AGENT INFECTING FORM. FINDINGS FROM A SYSTEMATIC REVIEW / Surtos de toxoplasmose humana e a forma infectante do agente. Achados de revisão sistemática

SUMMARY Toxoplasmosis, a worldwide highly prevalent zoonotic infection, is transmitted either by the oocysts, from water and soil, or the tissue cysts, in raw or undercooked infected meat, of Toxoplasma gondii. An ongoing debate is whether there are differences between the clinical and epidemiological characteristics of the outbreaks due to one or the other infective form of the agent. We performed a systematic review, recovering 437 reported outbreaks of which 38 were selected. They were complete reports containing ascribed Toxoplasma infecting form, and clinical and demographic data. There was no gender or age group selection in the outbreaks, which were described more often in the Americas. A large number of individuals were affected when oocysts, associated with soil and water contaminated with cat feces, were considered the transmission source. Onset of symptoms occurred early when the infection was ascribed to meat tissue cysts (11.4 ± 6.7 days) with sharpened temporal distribution of cases, while a broader and prolonged appearance of new cases was observed when oocysts in water were the source of the infection (20 ± 7 days, p < 0.001). Such information may be useful in the design and implementation of control strategies.

RESUMO Toxoplasmose, infecção zoonótica altamente prevalente no mundo, é transmitida pela ingestão de oocistos em água e solo ou cistos teciduais em carne crua ou mal cozida. Um debate em andamento é se há diferenças nas características clínicas e epidemiológicas de surtos devido a uma ou outra forma infectante do agente. Realizamos revisão sistemática a partir de 437 relatos de surtos da doença, selecionando 38 artigos completos que descreveram a forma infectante do Toxoplasmacom dados clínicos e epidemiológicos. Não houve seleção por gênero ou faixa etária nos surtos, descritos mais frequentemente nas Américas. Quantidade maior de indivíduos foi afetada quando oocistos, associados com solo ou água contaminados com fezes de gato, foram considerados a fonte de transmissão. O início dos sintomas ocorreu mais precocemente quando a infecção foi atribuída a cistos na carne (11,4 ± 6,7 dias) com distribuição temporal nítida de casos, embora um aspecto mais amplo e prolongado de novos casos foi observado quando oocistos na água foram a fonte de infecção (20 ± 7 dias, p< 0.001). Essas informações podem ser úteis no desenvolvimento e implantação de estratégias de controle.

Coccidia of gallinaceous meat birds in Brazil / Coccídios de galináceos de corte no Brasil

Coccidiosis is a disease that limits the production and marketing of gallinaceous birds in North America, especially quails, pheasants and chukar partridges. Virtually no research has been conducted in South America on the causative agents of diseases among these birds, including coccidia. The aim of this work was to make first observations on Eimeria spp. in the chukar partridge Alectoris chukar and the grey quail Coturnix coturnix, which are reared for meat in Brazil. Fecal and tissue samples were collected from commercial farms and were examined for oocysts, gross and microscopic lesions or endogenous stages. From this examination, it was found that partridges raised in Brazil did not have any visible infection. However, grey quails presented mild infection and two Eimeria species that had previously been described in other birds were identified.

A coccidiose é uma enfermidade limitante para a produção e comercialização de aves de corte na América do Norte, principalmente codornas, faisões e a perdiz de chukar. Praticamente nenhuma pesquisa foi realizada na América do Sul sobre os agentes causadores de doenças nessas aves, incluindo coccídios. O objetivo deste trabalho foi realizar as primeiras observações sobre Eimeria spp. em perdiz de chukar Alectoris chukar e codornas cinzentas Coturnix coturnix criadas para abate no Brasil. Amostras de fezes e de tecidos foram coletadas em granjas comerciais e examinadas para oocistos, lesões macroscópicas e microscópicas ou estágios endógenos. Após o exame, verificou-se que a criação de perdizes, no Brasil, não tinha infecção visível. No entanto, as codornas cinzentas apresentaram uma infecção leve e foram identificadas duas espécies de Eimeria descritas anteriormente em outras aves.

Prevalence of Cryptosporidium Infection among Inhabitants of 2 Rural Areas in White Nile State, Sudan

Cryptosporidium, a protozoan parasite that causes watery diarrhea, is found worldwide and is common in areas with low water hygiene. In February 2014, 866 stool samples were collected from the inhabitants of 2 rural areas in White Nile State, Sudan. These stool samples were assessed by performing modified acid-fast staining, followed by examination under a light microscope. The overall positive rate of Cryptosporidium oocysts was 13.3%. Cryptosporidium oocysts were detected in 8.6% stool samples obtained from inhabitants living in the area having water purification systems and in 14.6% stool samples obtained from inhabitants living in the area not having water purification systems. No significant difference was observed in the prevalence of Cryptosporidium infection between men and women (14.7% and 14.1%, respectively). The positive rate of oocysts by age was the highest among inhabitants in their 60s (40.0%). These findings suggest that the use of water purification systems is important for preventing Cryptosporidium infection among inhabitants of these rural areas in Sudan.

Differential Effect of Paromomycin and Nitazoxanide on Clinical Isolates of Cryptosporidia In Vitro.

Background: Cryptosporidiosis is one of the most difficult protozoan infections to treat, with only two drugs i.e. nitazoxanide and paromomycin known for treatment with variable response in different patients. Human cryptosporidiosis is accounted mainly by C. hominis and C. parvum. These two species or their subtypes are known to differ in clinical manifestations, and may differ in their response to drugs. So, we planned the study to see the effect of nitazoxanide and paromomycin on different isolates of Cryptosporidium in vitro. Methods: MDCK cell lines were used for in vitro growth of parasite and cytotoxicity of drugs to MDCK cells was determined by MTT assay after 3, 12 and 24 hours of drug exposure. Efficacy of non-toxic drug concentrations (<25% cytotoxic) on 12 Cryptosporidium isolates (7 C. hominis and 5 C. parvum) was determined at three different life cycle stages (in vitro growth, invasion and oocyst) by quantitative RT-PCR. Unpaired t-test was used to calculate the difference response of Cryptosporidium isolates to nitazoxanide and paromomycin. Results and conclusions: Cytotoxicity of nitazoxanide and paromomycin increased in dose and time dependent manner. After 24 hours of drug exposure, >25% cytotoxic effect was seen with nitazoxanide and paromomycin at concentrations of more than, 25μg/ml and 6mg/ml, respectively. Nitazoxanide was more effective than paromomycin in decreasing in vitro growth, invasion inhibition and reducing oocyst viability of Cryptosporidium isolates. Drugs effect was higher on growth inhibition followed by invasion inhibition and least in decreasing oocyst viability. Different isolates had variable response to drugs; cumulatively C. parvum isolates were more susceptible at particular drug concentrations than C. hominis isolates.

Fatal cryptosporidiosis in a calf / 대한수의학회지

A calf suffering from diarrhea was admitted to the Animal and Plant Quarantine Agency for diagnostic evaluation. Postmortem examination revealed that the mesenteric lymph node was enlarged and small intestine wall was thin. Microscopically, a large number of small round organisms were attached to the small intestine villi. Villous atrophy and proprial neutrophil infiltration were also observed. Based on modified Ziehl-Neelsen staining, electron microscopy, and ELISA results, the calf was diagnosed with fatal cryptosporidiosis.

Effects of Different Sizes of Glass Beads on the Release of Sporocysts from Eimeria tenella Oocysts

The oocyst wall is severed by means of mechanical injury or chemical agents. This study reports the percentage of in vitro sporocyst release following mechanical shaking in the presence of varying sizes of glass beads. Glass beads measured 0.5, 1, and 3 mm in diameter and were shaken with the oocysts for different times ranging from 5 sec to 5 min. Approximately 80% of sporocysts were released with 5 min of shaking in the presence of 3 mm glass beads, as well as 30 sec with 0.5 mm beads and 1 mm glass beads. The release of sporocysts of E. tenella was most efficient using 1 mm glass beads and treatment times of 30 sec to 1 min. Therefore, the use of 1 mm glass beads with 30 sec to 1 min of agitation is recommended in order to maximize sporocyst release and recovery and to improve the yield of viable sporozoites for use in biochemical, tissue culture, and immunological applications of coccidia.

A Waterborne Outbreak and Detection of Cryptosporidium Oocysts in Drinking Water of an Older High-Rise Apartment Complex in Seoul

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.

Compromised nutrition in gerbils infected by Cystoisospora felis detected through an animal performance analysis tool / Comprometimento nutricional em gerbis infectados por Cystoisospora felis detectado através de uma ferramenta de análise de desempenho animal

The impact of Cystoisospora felis infection on the nutritional efficiency of gerbils was studied. The variables weight gain and feed intake were measured during four weeks in 28 laboratory gerbils, of which 14 were inoculated with 3.5 × 10(5) sporulated oocysts of C. felis and the remaining 14 were controls. The animals from both groups were weighted, killed, eviscerated and had their carcasses and tissues weighted and compared. A modern tool designed for measuring nutritional performance of farm animals was applied. The results showed compromised nutritional efficiency of the infected animals within the first week after infection. The consequences of these results are discussed here, including the potential impact of infection on farm animals performance.

O impacto da infecção por Cystoisospora felis na eficiência nutricional de gerbis foi estudado. As variáveis ganho de peso e consumo de ração foram mensuradas durante quatro semanas em 28 gerbis de laboratório, dos quais 14 foram inoculados com 3,5 × 10(5) oocistos esporulados de C. felis e os 14 restantes serviram como controle. Os animais de ambos os grupos foram pesados, mortos, eviscerados e tiveram suas carcaças e tecidos pesados e comparados. Uma ferramenta moderna desenvolvida para mensurar o desempenho nutricional de animais de produção foi aplicada. Os resultados mostraram eficiência nutricional comprometida dos animais infectados na primeira semana após a infecção. As consequências destes resultados são discutidas aqui, incluindo o possível impacto de infecção no desempenho de animais de produção.

Study on the in vitro excystation of Cryptosporidium oocysts under different conditions / 中国人兽共患病学报

The aim of the present study was to evaluate the effect of storage conditions,pretreatment,temperature,time and excystation solutions on in vitro excystation of Cryptosporidium oocyst.Cryptosporidium andersoni oocyst was used as a model and the results showed that 0.5% sodium hypochlorite could enhance the excystation rates.But there was no significant difference compared with oocysts untreated by sodium hypochlorite(P＞0.05).0.75% synthetic sodium taurocholate and 1% bile solution could urge the excystation of oocysts,which were significantly different compared with the excystation rate of oocysts in 0.25% trypsin solution or in PBS(P<0.05).The excystation rates of oocysts in acidic water (pH =3) were similar with the rates in PBS (pH =7.2) but significantly different from the rates in alkaline water (pH =9) (P<0.01).Additionally,the excystation rate of oocysts in water of 24℃ was significantly lower than in water of 37℃(P<0.01),and the excystation rate of oocysts raised gradually at 37℃ with the passage of time.It's concluded that temperature,acidity and excystation solution were vital factors for the in vitro excystation of Cryptosporidium oocyst.A higher excystation rate could be observed when oocysts were pretreated with 0.5% sodium hypochlorite and treated with 0.75% synthetic sodium taurocholate at 37℃ for 3 hours.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Detection of Toxoplasma gondii oocysts in water: proposition of a strategy and evaluation in Champagne-Ardenne Region, France

Water is a vehicle for disseminating human and veterinary toxoplasmosis due to oocyst contamination. Several outbreaks of toxoplasmosis throughout the world have been related to contaminated drinking water. We have developed a method for the detection of Toxoplasma gondii oocysts in water and we propose a strategy for the detection of multiple waterborne parasites, including Cryptosporidium spp. and Giardia. Water samples were filtered to recover Toxoplasma oocysts and, after the detection of Cryptosporidium oocysts and Giardia cysts by immunofluorescence, as recommended by French norm procedure NF T 90-455, the samples were purified on a sucrose density gradient. Detection of Toxoplasma was based on PCR amplification and mouse inoculation to determine the presence and infectivity of recovered oocysts. After experimental seeding assays, we determined that the PCR assay was more sensitive than the bioassay. This strategy was then applied to 482 environmental water samples collected since 2001. We detected Toxoplasma DNA in 37 environmental samples (7.7 percent), including public drinking water; however, none of them were positive by bioassay. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method. Alternative methods can be used in conjunction with this one to determine the infectivity of parasites that were detected by molecular methods.

Fecal and Molecular Survey of Neospora caninum in Farm and Household Dogs in Mashhad Area, Khorasan Province, Iran

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dog is the definitive host for N. caninum and can infect dairy cattle. The aim of this study is to determine the prevalence of Neospora oocysts in feces of dogs from dairy farms. A total of 174 fecal samples was collected from 89 farm dogs and 85 household dogs during 2006 and 2008. Fecal samples of dogs were microscopically examined for detecting Hammondia Neospora-like oocysts (HNLO) by Mini Parasep(R)SF fecal parasite concentrator. HNLO were microscopically detected in 4 fecal samples (2.2%). The fecal samples with HNLO were examined by N. caninum-specific PCR. Two of the samples were positive for N. caninum. The 2 positive fecal samples were selected for inoculation to calves. Two inoculated calves were seronegative by ELISA for 4 months post-infection. This is the first report of finding N. caninum DNA in feces of farm dogs in Mashhad area, Iran.

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microliter were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.

Genotype and animal infectivity of a human isolate of Cryptosporidium parvum in the Republic of Korea

Cryptosporidium parvum oocysts were isolated from a child suffering from acute gastroenteritis and successfully passaged in a calf and mice (designated hereafter SNU-H1) in the Republic of Korea; its molecular genotype has been analyzed. The GAG microsatellite region was amplified by a polymerase chain reaction (PCR), with a 238 base pair product, which is commonly displayed in C. parvum. The isolate was shown to be a mixture of the genotypes 1 (anthroponotic) and 2 (zoonotic). To study its infectivity in animals, 2 calves and 3 strains of mice were infected with the SNU-H1; in these animals, the propagation of both genotypes was successful. In immunosuppressed (ImSP) BALB/c and C57BL/6 mice the number of oocysts decreased after day 10 post-infection (PI) ; but in ImSP ICR mice, they remained constant until day 27 PI. The results show that both the C. parvum genotypes 1 and 2 can be propagated in calves and ImSP mice.