Rumen ophryoscolecid protozoa in the hindgut of the capybara (Hydrochoerus hydrochaeris)

Rumen ophryoscolecid protozoa were observed in feces obtained from two capybara (Hydrochoerus hydrochaeris) housed at the Columbus Zoo, Columbus, Ohio. Total numbers were 58.1 X 10(4) and 19.0 X 10(4) per gram of wet feces in a male and female capybara, respectively. Four common rumen species of Entodinium were observed in the feces from both animals, with low numbers of Eudiplodinium maggii and Elytroplastron bubali also occurring in the male. Establishment of rumen ophryoscolecid ciliates in the intestinal tract of non-ruminant herbivores has not been reported previously.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Rumen Ciliates of Musk-Oxen (Ovibos moschatus Z.) from the Canadian Arctic1

Samples of rumen contents were obtained from 13 musk-oxen living on Banks Island, N.W.T., Canada. Total protozoan numbers ranged from 38.5 to 124.6 times 10⁴ per ml of rumen contents with a mean generic distribution of 52.4%Entodinium, 45.9%Diplodinium, and 1.7%Epidinium. A total of 18 protozoan species were found, six of which have not previously been observed in this host, i.e. D. (Diplodinium) costatum, D. (Ostracodinium) gracile, D. (Ostracodinium) trivesiculatum. D. (Eudiplodinium) medium, Epidinium quadricaudatum, and Epidinium parvicaudatum. Two new species of protozoa are described, Entodinium ovibos n. sp. and Diplodinium (Eudiplodinium) banksi n. sp.     

Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

Intestinal ciliate composition found in the feces of the Turk rahvan horse Equus caballus, Linnaeus 1758

Species composition and distribution of large intestinal ciliates were investigated in the feces from 15 Turk rahvan horses, living in the vicinity of Izmir, Turkey. Twenty-two ciliate genera consisting of 36 species were identified. This is the first report on intestinal ciliates in Turk rahvan horses and no previously unknown species were observed. The mean number of ciliates was 14.2 ± 13.9 × 10⁴ cells ml⁻¹ of feces and the mean number of ciliate species per host was 9.9 ± 7.1. No ciliates were observed in 2 horses. Bundleia and Blepharocorys were considered to be the major genera since these ciliates were constantly found in high proportions. In contrast, Paraisotricha, Didesmis and Gassovskiella were only observed at low frequencies. The ciliates found in this survey had almost the same characteristics as those described in previous reports, suggesting that there was no significant geographic variation in the intestinal ciliate fauna of equids.     

Rumen Ciliate Fauna of Some Brazilian Cattle: Occurrence of Several Ciliates New to the Rumen,Including the Cycloposthid Parentodinium africanum1

ABSTRACT. Parentodinium africanum was observed in rumen contents of four Brazilian cattle and constituted 4.4, 0.9, > 0.1, and 10.0% of the total ciliates. This appears to be the first observation of the family Cycloposthiidae in the rumen habitat. Blepharoconus krugerensis, previously observed in the intestines of the elephant, and an unknown species of ciliate with many characteristics similar to the family Paraisotrichidae were each observed in a single animal. A new subspecies, Diplodinium flabellum laterospinatum n. subsp., is also described. Total number of ciliates per ml of rumen contents ranged from 9.0 to 51.2 × 10⁴ and included an overall total of 55 species and 4 subspecies.     

Comparative studies of microbial populations in the rumen,duodenum, ileum and faeces of lactating dairy cows

Aims: Understanding factors that influence the composition of microbial populations of the digestive system of dairy cattle will be key in regulating these populations to improve animal performance. Although rumen microbes are well studied, little is known of the dynamics and role of microbial populations in the small intestine of cows. Comparisons of fingerprints of microbial populations were used to investigate the effects of gastrointestinal (GI) segment and animal on community structure. Methods and Results: Samples from four lactating dairy cows with ruminal, duodenal and ileal cannulae were collected. Terminal‐restriction fragment length polymorphism (T‐RFLP) comparisons of small subunit rRNA genes revealed differences in microbial populations between GI segments (P < 0·05). No significant differences in either methanogen populations or microbial community profiles between animals were observed. Quantitative PCR was used to assay relative changes in methanogen numbers compared to procaryote rRNA gene numbers, and direct microscopic counts were used to enumerate total procaryote numbers of the duodenal and ileal samples. Conclusions: T‐RFLP comparisons illustrate significant changes in microbial diversity as digesta passes from one segment to another. Direct counts indicate that microbial numbers are reduced by eight orders of magnitude from the rumen, through the abomasum, and into the duodenum (from c. 10¹² to c. 3·6 × 10⁴ cells per ml). Quantitative PCR analyses of rRNA genes indicate that methanogens are present in the duodenum and ileum. Significance and Impact of the Study: The contribution of microbial populations of the small intestine to the nutrition and health of cattle is seldom addressed but warrants further investigation.     

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 × 10⁹ and 3.54 × 10⁹ copies per milliliter for F. succinogenes, 5.10 × 10⁷ and 7.40 × 10⁷ copies per millilter for R. Flavefaciens, and 4.00 × 10⁶ and 6.00 × 10⁶ copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 × 10⁹, 4.55 × 10⁷, and 4.56 × 10⁶ copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.     

Experimental maduromycosis in the laboratory mouse

Summary 1. A 0.5 ml inoculum containing 1.4×10⁴ spores and/or mycelial fragments ofAllescheria boydii when injected intraperitoneally, did not cause death in 18–20 g female Swiss mice in 18 days.2. Focal, pin-point lesions developed in the liver and spleen of mice injected with the stock suspension and the 1:10 dilution of it (2.8×10⁴ and 2.8×10³ infective units ofA. boydii per ml, respectively); no lesions developed from the 1:100 dilution of the original suspension.3.Allescheria boydii was recovered from the livers and spleens of all mice, as well as from the peritoneal exudate of one mouse injected with the stock suspension ofA. boydii; the fungus was not isolated from heart's blood.4. Inability to cause death by the intraperitoneal injection ofA. boydii inocula might be attributed to the natural defense mechanisms in the mice or to the excessive dilution of the inoculum; it is improbable that any genetically controlled resistance to allescheriosis exists in the mice used in this study.Paper no. 664, Department of Botany and Plant Pathology, Ohio State University, 1735 Neil Avenue, Columbus 10, Ohio.This is the report of research done by the junior authors with the guidance of the senior author during a course in medical mycology in this department.     

Deoxyribonucleic acid homology among the species of the genus Bifidobacterium isolated from animals

Summary Representatives of the species of the genus Bifidobacterium isolated from bovine rumen, intestinal content of honey bees and pig feces were studied for their deoxyribonucleic acid similarities with DNA-DNA filter hybridization-competition experiments. The species were: B. asteroides, B. indicum and B. coryneforme from honey bees; B. ruminale and B. globosum from rumen; B. suis from pig feces; Mitsuoka's species B. thermophilum and B. pseudolongum. Strains of B. bifidum, B. infantis, B. longum and B. breve from human sources were included in some comparative experiments. The group of bacteria investigated is largely heterogeneous: virtually no homology exists among the species from rumen and pig feces; the species or types from bees, although unrelated with rumen and pig types, share with these a significant portion of the genome; the competitor DNA's from human strains did not at all react with any of the homologous systems. The annealing reaction was carried out under stringent conditions to ensure against mismatching of nucleotide sequences (70°C in Denhardt's reactive mixture). The separation within the genus Bifidobacterium of several specific entities is fully substantiated.This investigation was supported by a research grant of C. N. R. (Consiglio Nazionale delle Ricerche, Roma).     

An ultrastructural examination of the action of vinblastine on microtubules,neurofilaments and muscle filaments in vitro

Summary In vitro incubation of autonomic nerves with vinblastine sulphate (1×10^-4 M) caused a disappearance of microtubules within 15 min; during the following 15 min paracrystalline arrays appeared within the axons. An increase in the abundance of microfilaments was also observed, but these did not appear to arise from disaggregated microtubules since the increase in microfilament numbers was noted at an incubation time when crystal formation was extensive. Pretreatment of autonomic nerves with colchicine (2.5×10^-4 M) caused a reduction of approximately 80% in the numbers of microtubules, but did not prevent the formation of crystals on subsequent exposure to vinblastine. No ultrastructural changes were observed in myofilaments on incubation with vinblastine.We gratefully acknowledge the use of the electron microscopes in the Departments of Pathology and of Human Morphology, University of Nottingham School and thank Dr. Graham Robinson and Annette Tomlinson for their co-operation.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Capybara (Hydrochoerus hydrochaeris) distribution in agroecosystems: a cross-scale habitat analysis

Aim The aim of this study was to understand the spatial distribution of capybara (Hydrochoerus hydrochaeris) according to habitat attributes, using a multiscale approach based on fine‐ and broad‐scale variables in agroecosystems. Location Piracicaba river basin, south‐eastern Brazil (22°00′–23°30′ S; 45°45′–48°30′ W). Methods Potential habitats for capybara were selected in order to evaluate species presence/absence from October 2001 to December 2002. In each site, habitat attributes were sampled in the field (fine scale) and from GIS maps (broad scale) in terms of their presence or absence close to water. The variability of land cover between study sites was described by principal components analysis. Chi‐square tests were calculated for capybara presence/absence and the presence of each habitat attribute. A linear discriminant function analysis was used to describe to what extent the species’ presence could be explained by habitat attributes. Results The species presence was predominantly related to flat open areas (slope ranging from 0% to 6%) (χ² = 37.054, d.f. = 4, P < 0.001), covered by sugar cane or cultivated pasture (χ² = 84.814, d.f. = 9, P < 0.001). Terrain curvature, water meadows, aquatic vegetation, forest cover and open areas resulted in the best combination of variables, explaining 69.7% of capybara occurrence in the study sites in this river basin. Main conclusions Capybaras are widespread in the Piracicaba river basin, except in elevated areas. The spatial distribution of capybara was associated with the main types of land cover in the river basin – sugar cane plantations or pasture – both key food sources for capybara. This probably explains the species’ recent abundance in the region, since an intensive process of landscape alteration has taken place in this region owing to the expansion of agriculture in recent decades. These results may be useful in understanding the relationship between recent landscape modifications and the species’ population expansion in agroecosystems.     

Microbiological quality of pork meat from local Hong Kong markets

Over a 3-week period, samples of fresh, chopped, pork meat were taken every morning and afternoon from 50 meat stalls. Microbiological examination revealed that the samples had (c.f.u./g): total microbes, 1×10³ to 2.14×10⁶; mean probable numbers of coliforms and Escherichia coli, 1.51×10³ to 1.15×10⁴; and yeasts and moulds, 0 to 1.28×10⁴. Salmonella, found in 32 samples from 21 stalls, were serotyped as B (three samples), C1 (four) or E (25). No Campylobacter were found. Because microbial growth and/or contamination of the meat occurred during the day, samples taken in the afternoon had greater total counts (P<0.05) and contained detectable numbers of Salmonella more frequently (42% versus 22%) than those taken in the morning.The author is with the Department of Biology and Chemistry, City Polytechnic of Hong Kong, 83 Tat Chee Ave, Kowloon, Hong Kong     

Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10⁵ g⁻¹. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10⁶–10⁻¹⁰ g⁻¹). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

Sensitivity and Specificity of the Indirect Fluorescent Antibody Test in the Study of Four Murine Coccidia1

The sensitivity and specificity of the indirect fluorescent antibody (IFA) test for the detection of serum antibodies were examined in mice that were infected with Eimeria falciformis, E. ferrisi, E. papillata, or E. vermiformis. For the study of each species, five groups of mice were given graded inoculation doses of 10, 10², 10³, 10⁴, or 10⁵ sporulated oocysts in a primary infection. The sixth group was infected with three sequential doses of 1.5 times 10³, 1.5 times 10⁴, and 1.5 times 10⁵ sporulated oocysts per mouse at two- to three-week intervals. All groups of infected mice developed serum antibodies. Sera were titrated by the IFA test with purified sporozoites. Strong fluorescence and high IFA titers were observed with homologous reactions mainly with the sera from mice infected with the higher inoculation dose levels in primary infections and from those given three sequential inoculation doses. Immunological cross reaction among the four species of Eimeria occurred at dilutions of 1:10 to 1:160. Very weak or no fluorescence of free sporozoites was observed with sera from noninfected mice, and there was no fluorescence of sporozoites contained in intact sporocysts.