Prevalence and sequence diversity of Balantioides coli in pigs in Xinjiang, China.

Balantioides coli is a common intestinal parasitic protozoan in pigs. In the present study, 801 fecal samples of pigs from seven farms in Xinjiang were analyzed based on the ITS1-5.8S rRNA-ITS2 gene. The prevalence of B. coli was 4.2% (34/801), with the highest prevalence of 18.9% (18/95) occurring in Alaer, Xinjiang. B. coli was detected in all age groups (pre-weaned pigs, post-weaned pigs, fattening pigs and sows), with the highest rate in fatteners (6.9%, 9/129) and the lowest (1.2%, 2/169) in pre-weaned pigs. Significant differences (P = 0.000) were found among sampling sites but not among age groups (P = 0.084). Sequence analysis indicated that 34 sequence variants, including sequence type A (n = 11) and sequence type B (n = 23), occurred in all age groups. In this study, the existence of sequence type A suggested that B. coli poses a potential threat to human health. More studies are needed to better understand the distributions and public health significance of B. coli in China.

Metagenome of Gut Microbiota Provides a Novel Insight into the Pathogenicity of Balantioides coli in Weaned Piglets.

Balantioides coli plays an important role in the diarrhea of weaned piglets, but its pathogenic potential and interaction with gut microbes remain unclear. To investigate the impact of B. coli colonization on the gut bacterial structure and function of weaned piglets, a metagenomic analysis based on shotgun sequencing was performed on fresh fecal samples collected from ten B. coli-colonized piglets and eight B. coli-free ones in this study. The results showed that decreasing diversity and shifted composition and function of the bacterial community were detected in the weaned piglets infected by B. coli. In contrast to the B. coli-negative group, the relative abundances of some members of the Firmicutes phylum including Clostridium, Ruminococcus species, and Intestinimonas butyriciproducens, which produce short-chain fatty acids, were significantly reduced in the B. coli-positive group. Notably, some species of the Prevotella genus (such as Prevotella sp. CAG:604 and Prevotella stercorea) were significantly increased in abundance in the B. coli-positive piglets. A functional analysis of the gut microbiota demonstrated that the differential gene sets for the metabolism of carbohydrates and amino acids were abundant in both groups, and the more enriched pathways in B. coli-infected piglets were associated with the sugar-specific phosphotransferase system (PTS) and the two-component regulatory system, as well as lipopolysaccharide (LPS) biosynthesis. Furthermore, several species of Prevotella were significantly positively correlated to the synthesis of lipid A, leading to the exporting of endotoxins and, thereby, inducing inflammation in the intestines of weaned piglets. Taken together, these findings revealed that colonization by B. coli was distinctly associated with the dysbiosis of gut bacterial structure and function in weaned piglets. Lower relative abundances of Clostridiaceae and Ruminococcaceae and higher abundances of Prevotella species were biomarkers of B. coli infection in weaned piglets.

Question of agent of camel balantidiosis solved: Molecular identity, taxonomic solution and epidemiological considerations.

Domestic camels (Camelus bactrianus, the Bactrian camel; and Camelus dromedarius, the dromedary) are pseudo-ruminant herbivores kept as livestock in rural, inhospitable regions (cold deserts and dry steppes of Asia, arid to semi-arid regions of Africa, western and central Asia). Their close contact with humans makes them a potential reservoir for zoonotic parasite infections, as has been suggested for human balantidiasis. However, there is confusion about the ciliate species that infects camels: Infundibulorium cameli was originally described in dromedaries, but this name has almost never been used and most authors identified their findings as Balantioides coli and, to a lesser extent, Buxtonella sulcata, a cattle ciliate. To clarify the taxonomic status of the parasite and the corresponding zoonotic significance for camels, we performed morphological characterization of cysts and genetic analysis (SSU-rDNA and ITS markers) of B. coli-like isolates from Bactrian camels from Bulgaria and from dromedaries from Spain and the United Arab Emirates. Our results indicate that the camel ciliate is not B. coli, nor is it B. sulcata, but is a different species that should be placed in the same genus as the latter. Thus, camels are not a reservoir for human balantidiasis. Although the correct genus name would be Infundibulorium according to the principle of priority, this would lead to confusion since this name has almost fallen into disuse since its initial description, but Buxtonella is almost universally used by researchers and veterinarians for the cattle ciliate. We therefore propose to apply the reversal of precedence and use Buxtonella as the valid genus name. Consequently, we propose Buxtonella cameli n.comb. as the name for the camel ciliate.

Prevalence and risk factors of human Balantidium coli infection and its association with haematological and biochemical parameters in Ga West Municipality, Ghana.

BACKGROUND: In Ghana, Balantidium coli (B. coli) has been identified in vegetables and in pigs, although there is a paucity of data regarding human balantidiosis. This study sought to assess human B. coli infection in Ghana, factors associated with the infection as well as its association with haematological and biochemical parameters. METHODS: Two pig rearing communities in the Ga West Municipality, Ghana, were involved in this study. Stool and blood samples were collected from pig farmers and their exposed household members as well as relevant information on potential associated factors. Eosin-saline wet preparation was done on the same day of stool samples were collected while formol ether concentration technique was performed later. Haematological, biochemical parameters and serum electrolytes were determined using Celltac MEK-6500 K, PKL-125 biochemical analyser, and FT-320 electrolyte analyser, respectively. RESULTS: The overall prevalence of balantidiosis was 10.4 %, significantly higher among farmers (21.7 %) than in exposed household members (5.8 %) (x2 = 17.8, p = 0.000025). Of the 43 infected individuals, 20.9 % were co-infected with either Entamoeba histolytica, Giardia lamblia, or Schistosoma mansoni. In B. coli infection, mild to moderate anaemia together with a reduction in levels of platelet, albumin and, sodium, chloride, and bicarbonate ions were observed. However, white blood cells were significantly elevated in infected states. Poor farming practices such as free-range systems, improper disposal of pig faeces, lack of use of protective farming clothing, and unavailability of dedicated farming clothing were found to be associated with B. coli infection status. Finally, frequent diarrhea (OR = 12.30, p = 0.006) with occult blood (OR = 25.94, p < 0.0001) were found to be predictors of B. coli infection. CONCLUSIONS: Human balantidiosis is endemic in Ga West Municipality, Ghana. Individuals living closed to pig rearing communities presenting with frequent diarrhea with occult blood in stool should be screened and treated for balantidiosis to mitigate the clinical consequences of the infection.

Balantidiasis in humans: A systematic review and meta-analysis.

Balantioides coli is a protozoan that infects different hosts species, including humans, with zoonotic transmission. The parasite, which lives in the large intestine and in other organs, can lead to serious infections that may culminate in death. Information about human balantidiasis is generally still very scanty. In view of the above, the purpose of this study was to analyze the epidemiological, clinical and laboratory characteristics of human balantidiasis based on a systematic review and meta-analysis. The scientific articles were retrieved from various databases and were subjected to descriptive analyses, chi-squared tests, and summarized on a forest plot and the heterogeneity index (I2). A total of 103 articles were eligible and included in this review. Out of these 103 articles, 75 were clinical case reports and 28 were epidemiological studies, indicating a frequency of 997 (3.98%) people potentially infected with B. coli. The publication dates of the analyzed articles ranged from 1910 to 2020, but the majority (68.9%) were published between 1998 and 2020. A considerable number of these articles were published in South America and Asia, mostly in Brazil and India, respectively. However, in Africa, Ethiopia, was observed the higher number of infected people (47.5%). A significant association (p < 0.05) was identified between proximity to pigs and positivity for B. coli, since more than 16% infected people were in proximity with pigs and/or their excreta. Infection by the protozoan was classified mainly as intestinal, and the predominant symptom was dysentery. Extraintestinal infections were found in 27 individuals, with colonization of the genitourinary tract frequently highlighted. Direct examination (17.2%), followed by an association of direct examination and sedimentation (45.7%), were the most commonly performed parasitological techniques, and the most frequently diagnosed form was trophozoites, corresponding to 22.5% of cases. The most common treatment for parasitized individuals (11.8%) was an association of tetracycline drugs with nitroimidazole derivatives. The articles retrieved, mainly epidemiological ones, used in meta-analysis showed high heterogeneity (I2> 50%, p < 0.05), impairing the retrieval and comparison of results. Some articles were found to provide incomplete information, making it difficult to retrieve and analyze variables. However, this review enabled us to compile and restate factors that appear to be associated with cases of human balantidiasis.

Modified DMEM xenic culture medium for propagation, isolation and maintenance of Balantioides coli.

Balantioides coli is a known ciliated zoonotic protozoan that mainly causes diarrhea in humans and pigs. An efficient and reliable culture system for this parasite remains unavailable until now. In this study, a modified Dulbecco's modified eagle medium (DMEM) with pH 7.0-7.5, containing 5 mg/mL starch and 20% new calf serum, was optimized for propagation of B. coli at 28°C-32°C. At the growth-peaking stage, the average trophozoite density was up to 12,970 trophozoites per milliliter. A reproducible protocol for isolation and maintenance of this parasite was also developed based on the modified DMEM culture medium. Moreover, cloning results of B. colipopulations showed that 250 trophozoites in 3 mL modified DMEM medium were the minimal number of trophozoites that propagated to the growth-peaking stage, and finally obtained the individual population. However, less than 250 trophozoites failed to continuously grow in the modified DMEM culture medium under the optimal conditions for growth of B. coli. These data showed that the modified DMEM culture medium is an ideal and efficient medium for propagation and maintenance of B. coli in vitro and will help studies on its biology, genome, transcriptome, proteome, and drug screening.

Balantioides coli.

Balantioides coli (=Balantidium coli) is the only ciliate that parasitizes humans. Pigs are the main reservoir. Other species, as camels, cattle, donkey, sheep and goat have been also proposed as reservoirs for human infections. The parasite has a direct life cycle, being transmitted by the faecal-oral route. This type of cycle and the large number of host species imply an important potential for zoonotic transmission of the parasite. Infections are most commonly found in tropical and temperate regions, with prevalence up to 100% in pigs; high prevalence values have been also recorded in some non-human primates and camels. In humans, prevalence is usually under 10% in the population at risk. The main epidemiological factors involved in the transmission of this parasite include close contact with pigs, lack of basic sanitation infrastructures (water supply, wastewater disposal) and hygiene. Individual health status, intestinal microbiota and diet are also important for the onset of the infection. Outbreaks caused by this parasite are rare; those reported to date were related to poor hygienic conditions or to catastrophic natural disasters. Balantioides coli infections can be asymptomatic and symptomatic, which can be chronic (with intermittent diarrhoea), or acute (a dysenteric form which can be life-threatening). Efective treatments include tetracycline, iodoquinol and 5-nitroimidazole compounds (metronidazole, secnidazole). The main effective individual preventive measure is the use of disinfected water for drinking and other uses. Adequate water supply infrastructures, proper disposal of wastewater and animal faeces, and regular monitoring programs on farms will help limit transmission.

Alterations in the diversity and composition of gut microbiota in weaned piglets infected with Balantioides coli.

Balantioides coli is a ciliated parasite that can cause diarrhea when inhabiting the colon and cecum of pigs and humans. However, information regarding the changes in structure and composition of the gut microbiome in piglets infected with B. coli remains scarce. In this study, 16S rDNA amplicon sequencing was conducted on fecal samples from both B. coli-positive piglets with diarrhea and B. coli-negative piglets without diarrhea. The results showed that Firmicutes and Bacteroidetes were the major phyla in the two groups, and the relative abundance of the phylum Firmicutes, including Ruminococcaceae and Clostridiaceae, was significantly lower in the B. coli-positive samples than in the B. coli-negative ones (p < 0.05). Compared with B. coli-negative samples, Alloprevotella and Faecalibacterium showed distinctly higher relative abundances (p < 0.05) in the B. coli-positive samples, and the abundances of some potential pathogenic bacteria, including Escherichia-Shigella and Campylobacter, were positively correlated with B. coli infection and diarrhea of weaned piglets. In addition, there were statistically significant differences in fecal microbiota diversity and abundances of predicted functional genes between B. coli-colonized and B. coli-negative samples (p <  0.05). Taken together, these findings suggest that there were significant differences in the bacterial community composition, diversity, and functions between the B. coli-positive and B. coli-negative piglets, and the colonization by B. coli may be associated with the dysbiosis of gut microbiota structure in weaned piglets.

Prevalence and molecular identification of Balantioides coli isolates from pet guinea pigs in Central China.

Balantioides coli is the only known zoonotic ciliate that can infect humans and is usually acquired from swine. It has, however, been reported in other mammals, including guinea pigs, where infection prevalence and molecular characterization are relatively unknown. In the present study, 32 guinea pigs from two different pet markets in Luoyang city of the Henan province in China were evaluated for ciliate-like trophozoites or cysts by direct fecal smear microscopy. Positive samples were further characterized using 18S rDNA and ITS1-5.8S rDNA-ITS2 sequence analysis. Microscopy indicated that ciliate-like cysts were observed in the fecal samples of several guinea pigs, were spherical in shape, and exhibited sizes of 40-65 µm in diameter. The average cyst-positive prevalence in guinea pigs was 62.5%. Sequence analysis indicated that the guinea pig-derived ciliate isolates belonged to B. coli and included two genetic variants (A and B), of which genetic variant A was more dominant among the guinea pig samples. To the best of our knowledge, the present study is the first molecular identification of B. coli in guinea pigs and provides some important information for investigating the molecular epidemiology of B. coli.

Urinary balantidiasis in a patient with systemic lupus erythematosus and lupus nephritis: a case report.

BACKGROUND: Balantidium coli, a parasitic unicellular ciliate, often causes asymptomatic balantidiasis of the colon, but extraintestinal disease may occur rarely in immunosuppressed individuals. Renal balantidiasis associated with systemic lupus erythematosus has not been reported before. CASE PRESENTATION: We present a case of a 48-year-old Thai woman who presented with nephrotic syndrome due to systemic lupus erythematosus-related nephritis. Initially, few B. coli cysts were found in urine sediment, but these increased substantially following treatment with prednisolone. She made an uneventful recovery with 10 days of oral tetracycline therapy. No B. coli cysts were found in her stool. CONCLUSION: The route of infection in our patient was unclear but is likely to have been orofecal. Neither her infection nor its treatment caused a deterioration in her renal function.

Dysentery Caused by Balantidium coli in China.

Balantidium coli human infection predominantly occurs in tropical and subtropical regions in the world. Human case is extremely rare in China. This report details a case of B. coli infection in a 68-year-old man in China, who presented with history of abdominal pain, tenesmus, diarrhea with blood and was diagnosed as B. coli-caused dysentery. Our case indicates possible occurrence of Balantidium coli-related disease in cooler climates. This case is presented not only because of its rarity but also for future references.

Balantidium coli in domestic animals: An emerging protozoan pathogen of zoonotic significance.

Balantidium coli (B. coli) is an emerging ciliated protozoan parasite of zoonotic importance which causes a disease balantidiasis in a variety of host species including pigs, camels, ruminants, equines and even human. This disease has a cosmopolitan distribution with high prevalence rates in tropical and sub-tropical areas of the world due to favorable geo-climatic conditions for the development and survival of the parasite in these regions. Main reservoir hosts for this pathogen are pigs and animals; acquire infection mainly by the ingestion of the food or water contaminated with the porcine feces. The infected animal manifests clinical signs of anorexia, dehydration, profuse watery diarrhea and retarded growth. Wet mount slide prepared from intestinal scrapings and fecal material is used for the identification of trophozoites and cysts stages of this parasite. PCR can also be used to confirm the parasite. Secnidazole, oxytetracycline and metronidazole have varying efficacy against B. coli infection in various domestic animal species. There is no comprehensive literature available on the occurrence and distribution of the infection at international level. Therefore, the published data between 1989 and 2019 regarding this disease is critically analyzed to provide a detailed overview on this pathogen with special emphasis on geographical distribution of B. coli in domestic animals and different therapeutic agents used to treat this infection. This review will pinpoint the endemic regions which may be a source of potential disease outbreaks and will also help in application of more effectual control strategies against balantidiasis.

Neobalantidium coli: First molecular identification from the Eurasian wild boar, Sus Scrofa in Bushehr Province, Southwestern Iran.

Balantidium coli is a common parasite of pig and wild boars (Sus scrofa) which can infect humans and several species of mammals. This study aimed to determine the genotype of Balantidium isolated from Eurasian wild boars in Bushehr province, Southwestern Iran. Twenty-five faecal samples, originating from 25 wild boars captivated in our previous study, were processed. DNA was extracted from the faecal samples and PCR-amplified, targeting an ITS1-5.8s-rRNA-ITS2 region of Balantidium genome. PCR product was purified from the gel, and sequenced. BLAST analysis was performed in order to compare our isolates with other previously reported ones. A phylogenetic tree was constructed, using MegaX software, to find out the phylogenetic diversity of the isolates. With PCR it was possible to detect Balantidium DNA in the faecal samples of 13 out of 25 (52%) of the wild boars. BLAST analysis of seven isolates revealed that the isolates belong to the newly introduced genus Neobalantidium coli. Sequences of three isolates were deposited in the GenBank. Moreover, molecular analysis revealed six areas of nucleotide differences within the isolates and nine areas of difference between the sequences obtained in this study and those available in the GenBank. Phylogenetic analysis revealed that the sequences of isolates of this study have up to 2.2% dissimilarity from those published in the GenBank. The findings of this study, for the first time, revealed that some of the isolates of Balantidium originating from wild boars in Southwestern Iran belonged to the N. coli.

Endoparasitism of Greek ostriches: First report of Entamoeba struthionis and Balantioides coli.

Ostrich farming is a worldwide practice and an internationally developing industry. Among challenges in livestock production are the parasitic infections. The present study aimed to the investigation of the gastrointestinal parasites biota and prevalence in ostriches raised in different areas of Greece. A total of 141 clinically healthy ostriches originating from four different localities of Greece were coprologically examined for parasites of the gastrointestinal tract. Coprological examination revealed a considerably high rate of infection (65.9%) with protozoa; however, no helminths (trematodes, cestodes and nematodes) were detected. In detail, cysts of Entamoeba struthionis have been found in 57.4% of the examined birds. Moreover, 39.0% of sampled ostriches harboured cysts of Balantioides coli (syn. Balantidium coli), while oocysts of Cryptosporidium sp. were detected at a low percentage (2.1%). Partial sequences of the small subunit rRNA (16S rRNA) gene and the ITS region were amplified from pooled Entamoeba and Balantioides positive samples, respectively, confirming for the first time the presence of Entamoeba struthionis and Balantioides coli in ostriches in Greece. Some of these parasitoses require attention as they may affect productivity performance of the animals in commercial ostrich farming and possibly pose disease risk for livestock and humans.

[Colonic balantidiasis: report of a fatal case and review of the literature]. / Balantidiasis colónica: reporte de un caso fatal y revisión de la literatura.

Balantidiasis is a zoonosis produced by Balantidium coli, which inhabits the large intestine of the pig and man. Infection is uncommon in humans and mainly affects the colon. It occurs more frequently in developing countries, tropical and subtropical regions. Colonic balantidiasis can occur in most cases asymptomatically and reach in the most severe cases such as dysenteric diarrhea that can be complicated by low digestive bleeding and even perforation. We present the case of a 72-year-old man, from the Peruvian highlands, who was a farmer and breeder of swine and sheep, who came for 3 months of illness, initially characterized by liquid stools with bloodless mucus, abdominal pain, nausea, vomiting and in the last month of illness he presents dysenteric diarrhea. Colonoscopy was performed due to suspicion of infectious colitis, Balantidium coli trophozoites were found in the fresh sample and colonic tissue biopsy. Patient receives treatment with amebicide and antibacterial without clinical improvement, presenting as a complication multiple perforation in the sigmoid colon, treated with resection and terminal colostomy. Finally, the patient died despite receiving medical and surgical treatment.

Paromomycin sulfate is an effective treatment for balantidiasis in captive cynomolgus monkeys.

There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.

Diversity and prevalence of gastrointestinal parasites in two wild Galago species in Gabon.

In this study, we characterize the diversity and estimated infection levels of gastrointestinal parasites circulating in two galago species, Galago demidoff and G. thomasi in two sites situated in the Southeastern forests of Gabon. Our study reveals that eleven parasites including nine helminthes (Ascaris spp., Ankylostoma spp., Dicrocoelium spp., Gongylonema spp., Oesophagostomum spp., Lemuricola spp., Strongyloides spp. Trichostrongylus spp. and Trichuris spp.) and two protozoans (Balantidium spp. and Entamoeba spp.) may infect Galago spp. with high infection rates. The results show that: a very similar parasite spectrum is found in both host species; all the taxa identified were previously observed in other Primate species and/or Man. They also show that age, gender and forest type may influence infection rates and/or parasite diversity found in a particular host and/or geographic area.

The First Case of Diarrhoea in Tibetan Sheep, Ovis aries, Caused by Balantidium coli in the Qinghai Tibetan Plateau Area, China.

This study was carried out to determine the pathogen-causing diarrhoea in sheep Ovis aries in the Qinghai Tibetan Plateau Area, China. A trophozoite was identified as species of ciliate alveolates infecting the sheep based on morphological characteristics examined by microscope. It was mostly spherical, colourless and transparent, with many vesicles. Macronucleus and contractile vacuoles could not be distinguished. Size of the trophozoite was 80-180 × 70-150 µm and its surface was covered with cilia. Molecular analysis based on sequences of 18S rRNA and ITS genes confirmed the ciliate species as Balantidium coli. According to the literature, there have been many epidemiological investigations of B. coli infection in pigs, monkeys and humans. To our knowledge, this was the first report of B. coli infections in sheep in the Qinghai Tibetan Plateau Area of China, or eleswhere around the world. Importantly, the sheep case was rare but raised our concern that B. coli may spread across species and expand its host range.

Phylogenomic analysis of Balantidium ctenopharyngodoni (Ciliophora, Litostomatea) based on single-cell transcriptome sequencing.

In this paper, we present transcriptome data for Balantidium ctenopharyngodoni Chen, 1955 collected from the hindgut of grass carp (Ctenopharyngodon idella). We evaluated sequence quality and de novo assembled a preliminary transcriptome, including 43.3 megabits and 119,141 transcripts. Then we obtained a final transcriptome, including 17.7 megabits and 35,560 transcripts, by removing contaminative and redundant sequences. Phylogenomic analysis based on a supermatrix with 132 genes comprising 53,873 amino acid residues and phylogenetic analysis based on SSU rDNA of 27 species were carried out herein to reveal the evolutionary relationships among six ciliate groups: Colpodea, Oligohymenophorea, Litostomatea, Spirotrichea, Heterotrichea and Protocruziida. The topologies of both phylogenomic and phylogenetic trees are discussed in this paper. In addition, our results suggest that single-cell sequencing is a sound method of obtaining sufficient omics data for phylogenomic analysis, which is a good choice for uncultivable ciliates. The transcriptome data for Balantidium ctenopharyngodoni are the first omics data within the subclass Trichostomatia, and provide a good basis for ciliate phylogenomic analysis, as well as related omics analysis.

A Comparative Study of Four Methods for the Detection of Nematode Eggs and Large Protozoan Cysts in Mandrill Faecal Material.

Coproscopical methods like sedimentation and flotation techniques are widely used in the field for studying simian gastrointestinal parasites. Four parasites of known zoonotic potential were studied in a free-ranging, non-provisioned population of mandrills (Mandrillus sphinx): 2 nematodes (Necatoramericanus/Oesophagostomum sp. complex and Strongyloides sp.) and 2 protozoan species (Balantidium coli and Entamoeba coli). Different coproscopical techniques are available but they are rarely compared to evaluate their efficiency to retrieve parasites. In this study 4 different field-friendly methods were compared. A sedimentation method and 3 different McMaster methods (using sugar, salt, and zinc sulphate solutions) were performed on 47 faecal samples collected from different individuals of both sexes and all ages. First, we show that McMaster flotation methods are appropriate to detect and thus quantify large protozoan cysts. Second, zinc sulphate McMaster flotation allows the retrieval of a higher number of parasite taxa compared to the other 3 methods. This method further shows the highest probability to detect each of the studied parasite taxa. Altogether our results show that zinc sulphate McMaster flotation appears to be the best technique to use when studying nematodes and large protozoa.