Modified Ziehl-Neelsen Staining Research Articles

The incident and Molecular Identification of the Cryptosporidium Parvum in Cultivated Carp Fish (Cyprinus carpio) in Baghdad

The current study aimed to investigate the occurrence of Cryptosporidium species by traditional and molecular methods in freshwater fish in Baghdad city. A convenient number of stomach and intestinal continent samples were collected from three types of freshwater fish from different areas of Baghdad city from November, 2023 to April, 2024. All samples were examined phenotypically using a modified Ziehl-Neelsen stain and genotypically (nested PCR technique) based on a partial sequence of 18SrRNA genes with sequencing and phylogenetic tree analysis. The overall infection rate of fish with C. parvum was 5.5% (10/180). The common carp (Cyprinus carpio) was the only fish type infected, with a rate of 16.66% (10 60). January and February had the highest infection rate, 3.33%. The molecular identification and sequencing confirmed the infections with C. parvum. Molecular speciation of the parasite confirmed microscopical primary identification results. This study suggests that fish became a new source for transmission to humans and livestock.

Occurrence of Cryptosporidium Oocysts in Faeces, Milk and Water Sources in Sedentary Fulani Herds in Selected Local Government Areas of Kaduna State, Nigeria

Abstract Cryptosporidiosis is a neglected tropical zoonotic disease, commonly associated with a diarrhoea, caused by a protozoan parasite of the genus Cryptosporidium. The aim of this study was to determine the occurrence of Cryptosporidium oocysts in faeces, milk, and water sources, and its associated risk factors in sedentary Fulani herds. Seven hundred and fifty faecal samples (240, 180, 240 and 90 from cattle, sheep, goats and dogs, respectively), and 120 samples each, of milk and water were collected for this study. Faecal samples were concentrated by formol ether concentration technique, then stained using Modified Ziehl Neelsen staining method, to detect Cryptosporidium oocysts. Cryptosporidium oocysts in milk and water samples were also concentrated and stained with same stain. The prevalence of Cryptosporidium oocysts in (cattle, dog, sheep and goat faeces were 18.3 %, 15.6 %, 13.9 % and 4.2 %, respectively.), Significantly higher prevalence was observed in calves ≤ 1 year (P = 0.002), female cattle (P = 0.007) and animals with poor body condition (P < 0.001) than in the other groups. Goats with poor body condition scores had significantly higher prevalence (P = 0.008) than other groups while dogs ≤ 6 months of age also had significantly higher prevalence (P = 0.03) than older dogs. The prevalence of Cryptosporidium oocysts in bulk cow milk was 11.7 % while a prevalence of 13.3 % was observed in the water bodies sampled. Hence the findings of this study are of great public health significance, therefore, inhabitants of these LGAs should be informed and educated on the need for improvement of sanitary measures during the course of milking these animals, and the need for adequate pasteurization, of milk before consumption.

Prevalence and associated risk factors of Cryptosporidium infection in calves and hospitalized humans in Libo Kemkem, North Western Ethiopia.

Cryptosporidium infection is one of the major causes of acute gastroenteritis and diarrhoea caused by a protozoan parasite affecting vertebrates and humans. The disease is prevalent in cases of immunocompromised individuals. Despite the impact of the diseases in calf and hospitalized humans, well-documented studies are not available in the study area. The objectives of this study were to determine the prevalence of Cryptosporidium infection in calves and hospitalized humans and assess the major associated risk factors associated with Cryptosporidium infection in calves and hospitalized humans. A cross-sectional study was conducted from November 2020 to March 2021 on calf and human Cryptosporidium infection in Libo Kemkem District, North West Ethiopia. A total of 193 calves and 122 human stool samples admitted to the hospital were used for this study. Three kebeles were selected purposely, and individual calves were selected using a simple random sampling method. A number of sampled calves were allocated proportionally to the selected kebeles. Human samples were collected using a systematic random sampling method. Faecal and stool samples were examined using a modified Ziehl-Neelsen staining method. The overall prevalence of calf and human Cryptosporidium infection found in this study was 15.5% and 11.5%, respectively. Age of calf, breed, body condition, water source, faecal consistency and hygienic condition were found significantly (p<0.05) associated with Cryptosporidium infection in the calf. Similarly, the source of potable water, immunocompromisation and contact with domestic animals were found to be significantly (p<0.05) associated with Cryptosporidium infection in humans. There was a higher prevalence of Cryptosporidium infection in calves and humans in Libo Kemkem District. Therefore, the implementation of proper prevention methods of zoonotic Cryptosporidium infection between calf and human beings through significant risk factors is mandatory. Furthermore, additional studies to investigate the levels of economic importance of the disease should be conducted.

Bridging dermatology and hematology: a case of lepromatous leprosy with bone marrow involvement and pancytopenia.

Leprosy, caused by Mycobacterium leprae (M. leprae), primarily manifestswith cutaneous and peripheral nerve involvement. Systemic involvement, particularly in the bone marrow, is exceedingly rare. This report presents a case of lepromatous leprosy with bone marrow involvement, emphasizing the systemic nature of the disease and the importance of comprehensive diagnostic and management approaches. We aim to present a case of lepromatous leprosy with bone marrow involvement, detailing the clinical presentation, diagnostic evaluation, and management approach. A 65-year-old male with lepromatous leprosy and severe erythema nodosum leprosum developed pancytopenia. After undergoing comprehensive clinical evaluation, including history taking, physical examination, and laboratory investigations, bone marrow examination and molecular diagnostics using polymerase chain reaction (PCR) were performed to confirm the presence of M. leprae as an etiology for his pancytopenia. The bone marrow aspirate revealed hypercellularity with erythropoiesis and thrombopoiesis within normal limits. Foamy histiocytes with erythrophagocytosis were observed, along with the presence of M. leprae on Modified Ziehl-Neelsen stain. Molecular analysis confirmed M. leprae DNA in the bone marrow aspirate. Treatment with multi-drug therapy (MDT) and thalidomide resulted in normalization of blood counts and healing of skin lesions. This case underscores the systemic nature of leprosy and the rarity of bone marrow involvement, highlighting the importance of thorough evaluation in cases of persistent symptoms. Comprehensive diagnostic approaches, including bone marrow examination and molecular diagnostics, are essential for accurate diagnosis and timely initiation of appropriate treatment, ultimately improving patient outcomes and minimizing disease complications.

Survey of gastrointestinal coccidian parasites of pet birds in Tehran

ABSTRACT Birds of various families and genera comprise a notable proportion of veterinary clients in veterinary practice. Regarding the scarcity of information on avian coccidia in pet birds, this study was designed to investigate and identify the diversity of gastrointestinal coccidian protozoa in caged pet birds. Accordingly, droppings samples were collected in 2.5% potassium dichromate and examined for the presence of oocysts by wet smear and by flotation with the saturated salt solution. Detected oocysts were further morphologically specified. Special Modified Ziehl-Neelsen stain was used for the detection of Cryptosporidium spp. oocysts. Out of the 145 droppings that were collected for parasitological investigations, it was discovered that a Japanese quail (Coturnix japonica) had a Cryptosporidium sp. infection (1/145: 0.7%). Additionally, Isospora spp. was found in a common mynah and a canary (2/145: 1.4%), while Eimeria sp. was detected in a canary (1/145: 0.7%). Studied birds were clinically normal and no signs of intestinal infection were observed. The oocysts are described morphologically in detail and compared with the available data. Periodic faecal examination should be recommended for monitoring intestinal parasites in pet birds both from a public health perspective for Cryptosporidium and for avian host health in the case of non-zoonotic parasites. Additionally, the identification of parasite/host species composition will enhance our understanding of species diversity and the variety of hosts that coccidian parasites are capable of infecting.

Prevalence of Cryptosporidium spp. infection in rodents and chickens in Franceville, Gabon.

Cryptosporidium spp. members of the phylum Apicomplexa are obligate protozoan parasites capable of infecting various vertebrate hosts, including rodents and chickens. Infection caused by these parasites may lead to zoonotic diseases in humans. The aim of this study was to estimate the prevalence of Cryptosporidium spp. in rodents and domestic chickens sampled in Franceville, Gabon. Two hundred and eighty-five samples were collected, of which 185 samples were from rodents and 100 from domestic chickens. Microscopy after modified Ziehl-Neelsen staining and nested polymerase chain reaction targeting the small subunit (SSU) rRNA gene were used to examine Cryptosporidium spp. The overall prevalence of Cryptosporidium oocysts was 55.8%, with a prevalence of 72.4% in rodents and 25.0% in domestic chickens. Molecular analysis showed that Cryptosporidium spp. were present in 4.0% of the samples. No significant correlation was observed between Cryptosporidium spp. carriage and sex or location in this study. These results indicate that Cryptosporidium spp. persist and circulate in the studied animal species in Franceville, Gabon. Infection with Cryptosporidium is very common in rodents and chickens in Franceville. The potential risk of human contamination cannot be ruled out. More research should be conducted to characterize Cryptosporidium species circulating in rodents and chickens in Gabon. Such studies are essential to better understand the epidemiology of this protozoan and its potential impact on public health.

Prevalence of Cryptosporidium Infection among Children Attending Maryam Abacha Women and Children Hospital, Sokoto State, Nigeria

Study's Excerpt/Novelty This study investigates the prevalence of Cryptosporidium among children attending Maryam Abacha Women and Children Hospital, Sokoto, using Formol ether concentration and Modified Ziehl Neelsen staining methods. It identifies a high prevalence of (71.1%) and shows a strong correlation between Cryptosporidium infection, diarrhea, and source of drinking water. These findings underscore the necessity for routine diagnostic testing of Cryptosporidium among children for improved detection, treatment, and overall patient outcomes in the region. Full Abstract Cryptosporidium infection is ranked as the second most common cause of diarrheal disease and fatalities among children in developing nations. Nonetheless, due to the fact that many healthcare providers do not consistently request Cryptosporidium diagnostic tests, the prevalence of cryptosporidiosis is probably underestimated. The current study is therefore aimed at investigating the prevalence of cryptosporidiosis among children attending Maryam Abacha Women and Children Hospital (MAWCH), Sokoto. During the timeframe of April to June 2021, stool samples were collected from 114 diarrheic and non-diarrheic children and analyzed using Formol ether concentration and Modified Ziehl Neelsen staining methods. Out of 114 samples analyzed, 81(71.1%) were positive for Cryptosporidium oocysts. A higher prevalence of Cryptosporidiosis was observed in males 45(39.5%) compared to females 36(31.6%) participants and higher 41(36.0%) in younger children aged 0-1 years than the older children. Similarly, a greater prevalence of Cryptosporidiosis was observed in diarrheic children (42.1%) than in non-diarrheic children (28.9%). A statistically significant relationship was observed between the occurrence of Cryptosporidium and diarrhea (P = 0.002) and the source of drinking water (P = 0.001). Routine diagnosis of Cryptosporidiosis among children is vital and can enhance effective diagnosis, treatment, and consequently, improvement in the patients' well-being.

Occurrence and factors associated with Cryptosporidium infection in livestock in three districts of Zambia

Cryptosporidium is one of the most important enteric diarrhoeal parasites that infect humans and animals worldwide. The current study investigated the occurrence and risk factors associated with Cryptosporidium infection in ruminants aged ≤6 months in Monze, Mumbwa, and Lusaka districts of Zambia. Faecal samples were collected from 328 calves, 190 lambs, and 245 goat kids and analysed for Cryptosporidium oocysts using modified Ziehl Neelsen staining. A closed structured questionnaire was used to obtain epidemiological characteristics and potential risk factors for Cryptosporidium infection. The overall occurrence of Cryptosporidium was 7.9% (60/763), while that in calves, lambs and goat kids was 14.5% (47/328), 5.3% (10/190), and 1.2% (3/245) respectively. Watery/pasty stool and sampling during the rainy season were independently associated with increased risk of infection. In calves, the odds of infection increased during the rainy season, while daily kraal cleaning reduced the infection risk. Lambs showed increased odds of infection with pasty/watery stool and male sex, whereas the wearing of protective clothing by handlers significantly reduced the risk. There were district variations in infection occurrence with Mumbwa district having higher prevalence. The findings of this study show that livestock in Zambia continue to be frequently infected with Cryptosporidium. Protective measures and appropriate farm cleanliness should be implemented in control of this infection. Regional and host-species-specific variations emphasize the need for targeted interventions. These findings, therefore, contribute to effective strategies for Cryptosporidium control, promoting good livestock health and management.

Incidence of Cryptosporidium: A Neglected Parasite in Immunocompetent Adults in Swabi

Objective: To highlight the increasing incidence of Cryptosporidium in immunocompetent adults and the resultant altered epidemiology. Methodology: This study was conducted over a period of one year, from 1st January 2023 to 1st of January, 2024 at Microbiology Laboratory MTI- GKMC/BKMC, Swabi. Modified Ziehl Neelsen staining was performed on the received stool samples from patients presenting with wide range of symptoms associated with Irritable Bowel Syndrome (IBS). Abdominal pain, recurrent episodes of diarrhea and weight loss were the main symptoms. Modified ZN stain being one of the most reliable, quick and cost effective methods for the detection of cysts of Cryptosporidium using 40X and oil immersion lens was used for diagnosis. Results: A total of 62 samples were received from immunocompetent patients in the said period out of which 34% were positive for the oocysts of Cryptosporidium species. Out of a total of 21 positive samples 62% were males and 38% were females. The age range of the patients spanned between 20 to 60 years. Conclusion: Knowing this high incidence 34% of Cryptosporidium spp. in the healthy adults, necessitates to prioritize steps for the prevention of this infection by implementation of public health measures.

Cryptosporidium and associated risk factors in local breeds of pigs kept in Donga Local Government Area Taraba State, Nigeria

Cryptosporidium, an enteric protozoan parasite, poses economic losses in animal production and has zoonotic potential. This study aimed to determine the prevalence of cryptosporidium and associated risk factors in local pig breeds within Donga Local Government Area of Taraba State, Nigeria. A cross-sectional study was conducted, and a total of 384 faecal samples were collected from pigs in eight sampling sites. The samples were examined using the modified Ziehl-Neelsen staining technique.The results showed an overall prevalence of 27.9% (107/384) for cryptosporidium oocysts. The prevalence was significantly higher in pre-weaned piglets (64%) compared to post-weaned (26.9%) and adult pigs (24.14%). Female pigs had a significantly higher prevalence (49.21%) than males (17.4%), and diarrhoeic pigs had a significantly higher prevalence (55.2%) than non-diarrhoeic (22.9%). The prevalence was higher in the rainy season (42.4%) than in the dry season (10.3%).The detection of Cryptosporidium spp. oocysts in the faeces of pigs suggests that these animals may serve as potential reservoirs and shedders of this protozoan parasite, contaminating the environment and serving as a source of infection to other animals and humans. Adequate personal and environmental hygiene, along with prompt veterinary care, should be intensified.

Identification of major protozoal enteropathogens causing calf diarrhea in dairy farms in and around Holeta Town, Oromia Special Zone, Ethiopia

Calf diarrhea can be caused by a variety of pathogens, including bacteria, viruses, protozoa, and intestinal parasites. Giardia, Eimeria, and Cryptosporidium are the most significant protozoan parasites and are all individually and ollectively infectious. A case-series study was carried out in and around Holeta dairy farms, Oromia Special Zone, Ethiopia, from November 2017 to April 2018 to identify the main protozoan enteropathogens from diarrheic calves. Samples were purposely collected from three dairy farms: Serkalem Dairy Farm (SDF), Holeta Agricultural Research Centre (HARC), and Ada Berga Agricultural Research Centre (ABARC). A total of 93 fecal samples were taken from diarrheic calves of up to 4 months of age. Protozoan enteropathogens were identified using flotation and modified Ziehl Neelsen staining methods. Cryptosporidium, Eimeria, and Giardia were detected in 44 (47.3%), 50 (53.8%), and 34 (36.6%) of the diarrheic fecal samples examined, respectively. The findings indicated that there were 17 (18.27%) cases of Cryptosporidium, Eimeria, and Giardia as mixed infections, 16 (17.2%) cases of Cryptosporidium and Eimeria, 5 (5.4%) cases of Cryptosporidium and Giardia, and 4 (4.3%) cases of Eimeria and Giardia, compared to 13 (13.97%), 6 (6.45%), and 5 (5.4%) cases of Cryptosporidium, Eimeria and Giardia as single infections, respectively. The selected farms had significant prevalence levels of three common protozoa: Eimeria, Giardia, and Cryptosporidium. Further molecular research is required to identify the species and genotype levels of protozoal enteropathogens and related risk factors.

Intestinal Parasitic Infections in People Referring to the Central Laboratory of Meshkin Shahr County, Ardabil Province, Iran.

Intestinal parasitic infections are still a considerable global public health problem. We aimed to determine the frequency of intestinal parasitic infections among people referring to the central laboratory of Meshkin Shahr City, Ardabil Province, Iran. In this cross-sectional survey, 460 fecal samples were collected randomly from persons referred to the central laboratory of Meshkin Shahr City, from January to June 2022. The samples were examined by direct wet-mount, Trichrome and modified Ziehl-Neelsen staining, formalin ethyl acetate sedimentation, and agar plate culture. The frequency of intestinal parasites was 15.7% (72 out of 460 cases), with some people with numerous intestinal parasites. The frequency of protozoan infections (13.9%) was higher than the helminthic infections (2.6%). Blastocystis spp. (8.1%) was the most prevalent detected intestinal protozoan. Entamoeba coli (5.7%), Dicrocoelium dendriticum (2.2%), Giardia lamblia (1.5%), Fasciola spp. (0.2%), and Hymenolepis nana (0.2%) were other detected parasites. In- spite of betterment of the health condition in Iran and reduction of parasitic infection, intestinal parasitic infections are still a considerable public health issue in some parts of Iran.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1β and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1β and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Prevalence and risk estimates of Cryptosporidium oocysts infection associated with consumption of raw-eaten vegetables in Maiduguri metropolis LGAs, Northeast Nigeria

Cryptosporidium is one of the most important protozoan parasitic pathogens, and it is a common cause of diarrhoea in humans, domestic animals, and wild vertebrates and has serious public health threats. A cross-sectional study was designed to determine the prevalence of Cryptosporidium oocysts in raw-eaten vegetables in Maiduguri Metropolitan Council (MMC) and Jere Local Government Areas (LGAs). A total of 400 samples were collected from four (4) different locations, namely Tashan Bama, Gomboru, and Monday Markets (n = 100), while fifty (n = 50) each from 202-Vegetable-Vendors and Unimaid Commercials. A total of 16 visits were conducted in all the sampling areas (twenty-five samples per visit). The Cryptosporidium oocysts were detected using the Modified Ziehl–Neelsen Staining Technique. The locations, sources, and types of raw-eaten vegetables were also assessed. The oocysts were confirmed (100×) as bright pink spherules. Data generated were analyzed using IBM-SPSS V23.0, and p ≤ 0.05 was considered significant. Out of the total samples (n = 400) analyzed, cabbage appeared to have the highest number of 10 (12.5%) of Cryptosporidium oocysts detected, while Tomato and garden egg had 9 (11.3%) and 1 (1.2%), respectively. There was a statistically significant association (χ2 = 12.5, P = 0.014) between the presence of Cryptosporidium oocysts in raw-eaten vegetables and vegetable types. Among the sources of the vegetables sampled, Alau had the highest number of Cryptosporidium oocysts, 15 (12.5%), followed by Kilari-Abdullahi and Zabarmari sources with 4 (10.0%) and 4 (5.0%), respectively. However, Jetete appeared to have the least number 2 (2.5%) of oocysts, and there was a statistically significant association (χ2= 10.4, P = 0.034) between the presence of Cryptosporidium oocysts and the sources of vegetables and fruits. The study concludes that the raw-eaten vegetables sampled from Maiduguri Metropolis were contaminated with Cryptosporidium oocysts. The study recommends that all raw-eaten vegetables should be from cleaned sources and washed before consumption. Consumers should be enlightened on the hygienic measures in the food chain in line with the Hazard Analysis and Critical Control Points (HACCP) principles.

Enteric Protozoan Parasitosis and Associated Factors among Patients with and without Diabetes Mellitus in a Teaching Hospital in Ghana.

Enteric protozoa infections (EPIs) could worsen clinical outcomes in patients with diabetes mellitus and therefore requires prompt and accurate diagnosis and attention. This study aimed to determine the burden of EPIs and their associated factors among patients with and without diabetics at the Komfo Anokye Teaching Hospital (KATH) in Ghana. Again, the diagnostic performance of parasitological techniques routinely used for diagnosis was assessed. A total of 240 participants (made up of 140 patients with diabetes and 100 patients without diabetes) were recruited into the study by simple random sampling from November 2020 to May 2021. Stool samples of participants were collected, along with their demographic information, and examined using the saline direct wet mount (DWM), formol-ether concentration (FEC), and modified Ziehl-Neelsen staining (ZNS) techniques for the presence of enteric protozoans. Enteric protozoa were found among 62/140 (44.3%) diabetic patients and 13/100 (13.0%) nondiabetic patients. The predominant protozoa identified were Cryptosporidium spp. (17.86%) among patients with diabetes and Blastocystis hominis (7.0%) among patients without diabetes. EPI was associated with diabetes mellitus status (AOR = 3.48, 95% CI, 1.55-7.79), having diabetes for more than five years (AOR = 3.83, 95% CI, 1.65-8.86) and having comorbidity (AOR = 2.93, 95% CI, 1.33-6.45). The FEC technique had the highest sensitivity (100.0%), specificity 94.3% (95% CI, 91.35-97.22), and accuracy 95.0% (95% CI, 88.54-98.13) when compared to other techniques for diagnosis. EPIs are a significant health problem among patients with diabetes at KATH, and therefore antiparasitic drugs should be included in their treatment protocols for better health outcomes. Again, the FEC technique has demonstrated better performance in detecting EPIs and is therefore recommended to achieve early and accurate diagnosis of EPIs.

Detection and molecular characterization of Cryptosporidium spp. in dairy calves in Lisbon and Tagus Valley, Portugal

Cryptosporidium is a protozoan parasite with worldwide distribution, infecting a wide range of hosts with some zoonotic species. Calves have been identified as one of the most common reservoirs of this parasite. However, little is known about the genetics of Cryptosporidium in calves in Portugal. This study aimed to molecularly characterize infections of Cryptosporidium in pre-weaned calves from the Lisbon and Tagus Valley (LTV) in Portugal. Fifty-two samples were collected from calves from eight dairy and two beef farms in LTV, Portugal. Cryptosporidium oocysts were detected by Modified Ziehl-Neelsen staining (MZN) and direct immunofluorescent assay (DFA). MZN and DFA revealed the presence of Cryptosporidium oocysts in 40.4% (21/52) and 67.3% (35/52) samples, respectively. Positive samples were analyzed by PCR-RFLP of the 18 s rRNA gene for species identification. DNA amplification of the 18S rRNA gene was successful for 88.6% (31/35) of samples. Cryptosporidium parvum was identified in 96.8% (30/31) of the samples, and from one sample Cryptosporidium bovis was identified. Cryptosporidium parvum positive samples were subtyped by sequencing the PCR product of a partial fragment of the 60 kDa glycoprotein (gp60) gene. Subtype analysis of the C. parvum isolates revealed that all isolates belonged to subtype family IIa. Four subtypes were recognized within this subtype family, including the hyper-transmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (27/30), IIaA14G2R1 (1/30), IIaA16G2R1 (1/30) and IIaA19G2R1 (1/30). To our knowledge, this is the first report of C. bovis, and C. parvum subtypes IIaA14G2R1 and IIaA19G2R1 in cattle in LTV, Portugal. The presence of the zoonotic C. parvum subtype in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, highlighting the importance of animal-to-human infection transmission risk. Further molecular studies are required to better understand the epidemiology of cryptosporidiosis in Portugal.

Molecular Characterization of Cryptosporidium Oocysts in under-Five Children with Diarrhoea in Ile-Ife, Nigeria

Aim: Cryptosporidiosis is the fifth leading cause of diarrhoea-related death globally among under-five children. This study was conducted to identify and characterize oocysts of Cryptosporidium in under-five children with diarrhoea in Ile-Ife.&#x0D; Study Design: It was a case-control study.&#x0D; Place and Duration of the Study: Department of Medical Microbiology and Parasitology, Obafemi Awolowo University between April, and October 2019.&#x0D; Methodology: Ethical approval was obtained for this study. Consent was also obtained from the parents of the children. Stool samples from 53 children with diarrhoea (cases) and 94 children without diarrhoea (controls) under five years of age were collected. A structured proforma was used to obtain relevant information from the parents of the children. Stool samples were collected and examined macroscopically for consistency, appearance, and colour. Modified Ziehl-Neelsen staining was carried out on all the stool samples. The samples positive for Cryptosporidium oocysts by staining were genotyped by nested polymerase chain reaction and confirmed by sequencing of their 18S rRNA genes. Data generated were analyzed using descriptive and inferential statistics with SPSS software version 20 and STATA 15. &#x0D; Results: The prevalence of cryptosporidiosis among the study participants was 23.1% with rates of 43.4% (23) and 11.7% (11) for diarrhoeic and non-diarrhoeic children respectively. Of the 34 stool samples analysed, 6 (17.6%) were amplified by nested PCR and 4 (11.8%) were identified by sequencing. The species of Cryptosporidium identified were Cryptosporidium parvum (75%) and Cryptosporidium bovis (25%).&#x0D; Conclusion: In conclusion, Cryptosporidiosis is prevalent among under-five children with Cryptosporidium parvum and Cryptosporidium bovis as the infecting species.

Ocorrência de Cryptosporidium spp. em bezerros de rebanhos leiteiros do norte de Antioquia, Colômbia

ABSTRACT Cryptosporidium spp. are important enteropathogen protozoan parasites that infect humans and other animals throughout the world. Cryptosporidiosis in cattle industry leads to considerable economic losses due to diarrhea, dehydration, growth retardation, weight loss, and possibly death, however, data on the occurrence ofCryptosporidiumspp. in cattle in Colombia are limited. Therefore, this study aimed to investigate the occurrence and possible factors associated to the excretion of Cryptosporidium spp. oocyst in pre-weaned calves from dairy farms in Northern Antioquia, Colombia. In addition, Sheather’s sugar floatation (SSF), and Modified Ziehl-Neelsen staining (MZN) methods were compared. A total of 41 fecal samples were collected from calves between 1 and 92 days of age of which 23 were positive (56.1%). Crossbreed calves were nine times less susceptible than purebred dairy cattle to excretion of Cryptosporidium spp. oocysts (OR=0.10). MZN was the best technique for the detection of oocysts in fecal samples, however, the mean number of days to detect cryptosporidial oocysts was lower for the SSF method. Cryptosporidium is widespread among calves under 2 months of age in dairy herds of Northern Antioquia, although further investigations considering a greater number of farms and animals are necessary.

Dynamics of protozoal excretion in the faeces of calves during the first 28 days after arrival at the fattening farm indicate infection before regrouping and show poor temporal correlation with diarrhoea

BackgroundCalves in dairy cattle production in Switzerland are transported to a fattening farm at the age of 3–5 weeks, and frequently suffer from diarrhoea within the first 14 days after arrival. To characterise the role of intestinal protozoa in this, we investigated the excretion dynamics of Eimeria, Cryptosporidium and Giardia during the first 28 days after the arrival and regrouping of calves at fattening farms.MethodsA total of 610 faecal samples from 122 calves (mean age 37.3 days; mean body weight 79.8 kg) were collected on seven different fattening farms during the first 28 days after the arrival and regrouping of the animals. The farms were visited between January and April (cold season; n = 4) and between June and August (warm season; n = 3). The samples were collected rectally on days 1, 4, 7, 14 and 28, assessed for consistency, and analysed using the McMaster method for quantitative determination of the number of Eimeria oocysts per gram of faeces (OPG), flotation for morphological differentiation of the unsporulated Eimeria oocysts, a concentration method for the semi-quantitative determination of Giardia cysts, and modified Ziehl–Neelsen staining for semi-quantitative determination of Cryptosporidium oocysts.ResultsOverall, 50.8% (62/122) of the animals had diarrhoea during the study period. However, the faecal excretion of protozoal pathogens was neither associated with diarrhoea nor with body weight gain of the animals. Altogether, 90.2% (110/122) of the calves were Eimeria positive. Eimeria zuernii was excreted by 51 (41.8%) and Eimeria bovis by 68 (55.7%) animals. In the warm season more animals tested positive for Eimeria and OPGs were higher than in the cold season. There was no correlation between the age of the calves and the OPG values. Overall, 64.8% (79/122) of the calves excreted Eimeria oocysts within the first 7 days, indicating that they had been infected with the parasite on the dairy farm of origin. Eighty-nine calves (73.0%) excreted Giardia cysts, with more positive animals in the cold (80.3%) compared with the warm season (64.3%). Only Giardia duodenalis assemblage E was identified. Cryptosporidium oocysts were microscopically detected in 14 animals (11.5%) on five farms. Cryptosporidium spp. were present in a total of 12 animals, i.e. Cryptosporidium parvum in nine, Cryptosporidium ryanae in two, and Cryptosporidium bovis in one animal.ConclusionsA better understanding of the temporal dynamics of protozoal infections in calves is helpful for the implementation of appropriate measures to protect the health of these animals at a critical phase in their lives. Our results indicate that factors other than those examined in the present study contributed to the onset of diarrhoea in the calves.Graphical abstract

Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl-Neelsen Stain.

(1) Background: The wet mount staining technique is a simple, economical, and rapid method for detecting parasitic stages. The objective of the current study was to evaluate wet mount diagnostic stains for Cryptosporidium infection in human faecal samples and to compare the sensitivity and qualitative performance of toluidine blue (TolB) and modified Ziehl-Neelsen (mZN) stain. (2) Methods: The collection, purification, and molecular amplification of Cryptosporidium oocysts were performed. TolB, malachite green, trypan blue, and crystal violet were evaluated qualitatively to diagnose Cryptosporidium oocysts. The outperforming stain was compared to mZN using a quantitative evaluation and qualitative scoring system. (3) Results: The oocysts of Cryptosporidium parvum were effectively purified and utilized for spiking. TolB was the most effective diagnostic stain among wet mount stains for detecting Cryptosporidium oocysts. TolB outperformed mZN in terms of sensitivity, with 100% versus 79% at a concentration of 104 and 49% versus 23% at a concentration of 102. TolB had the most favourable qualitative stain characteristics against mZN; however, sample freshness and being a temporary stain were crucial considerations. (4) Conclusions: This study emphasizes that TolB, as a routine wet mount technique for screening Cryptosporidium infection, will provide a more sensitive and faster method than mZN staining.