A virulent mono-eukaryotic culture of Histomonas meleagridis is capable of inducing fatal histomonosis in different aged turkeys of both sexes, regardless of the infective dose

Histomonosis (syn. histomoniasis) is a parasitic disease which affects predominately turkeys but also other avian species. Concurrent with the ban of therapeutic and prophylactic substances, the disease, caused by the flagellated protozoon Histomonas meleagridis, is more frequently reported. Due to somewhat diverse results reported in the past, a well-characterized culture was used in the present study to investigate the possible influence of certain parameters on the outcome of the disease. For this study, turkeys were infected with different doses of the mono-eukaryotic culture Histomonas meleagridis/Turkey/Austria/2922-C6/04 using birds of both sexes at various ages. All study groups consisted of 14 birds, of which 10 birds were directly infected via the cloacal route and four birds were kept as in-contact birds. This scheme was used to investigate the pathogenicity of the cloned isolate in 1-day-old and 14-day-old turkeys. In 8-week-old turkeys, only eight birds out of 12 were infected. When 1-day-old and 8-week-old turkeys were infected with 10(4) histomonads per bird, all turkeys died between 11 and 21 days postinfection or had to be euthanatized due to their poor condition. In a group of 14 poults, infective doses of either 10 histomonads (100 histomonads among 10 birds) or 10(3) histomonads per bird had hardly any influence on the first notification of clinical signs. However, even though the onset of clinical signs and mortality was delayed with the lower dose, none of the birds survived the infection. As a consequence, no differences were noticed between male and female turkeys using the mono-eukaryotic culture of Histomonas meleagrigis/Turkey/Austria/2922-C6/04 in the current experimental setting.     

Progression of histomonosis in commercial chickens following experimental infection with an in vitro propagated clonal culture of Histomonas meleagridis

Four commercial strains of chickens, namely, ISA brown leghorn (ISA), TETRA-SL brown (TETRA-SL), Lohmann brown (LB), and Lohmann LSL (LSL), were infected with a well-defined clonal culture of Histomonas meleagridis (H. meleagridis/Turkey/Austria/2922-C6/04) to investigate their susceptibility to histomonosis. Each group included 16 chickens, which were housed under the same conditions in separate pens. All chickens were infected with 10(4) histomonads orally and intracloacally at 14 days of age. No mortality or clinical signs were observed during the experiment in all birds. Three birds of each chicken strain were euthanatized on days 4, 7, 10, 14, and 21 postinfection. Incidence of histomonosis on the basis of cecal lesions was found to be 64.00% in TETRA-SL, 62.50% in LB, 53.12% in LSL, and 43.75% in ISA chickens. Fewer lesions were noticed in livers than in ceca, with an incidence of 15.62% in TETRA-SL, 9.37% in LB, and 3.12% in ISA chickens. No liver lesions were found in the LSL chickens. Statistical analysis revealed that there was no significant difference (P > 0.05) in susceptibility to experimental H. meleagridis infection based on cecal and liver involvement. Polymerase chain reaction (PCR) and immunohistochemistry were found to be reliable tools to confirm the presence of histomonads and changes in the ceca. However, some negative PCR results were recorded from the livers despite the presence of macroscopic lesions. Additionally, DNA of H. meleagridis was detected by PCR in a few of the lungs, but immunohistochemistry was negative. Nucleic acid of the protozoan parasite was not detected in samples from kidney, brain, spleen, or bursa of Fabricius. Altogether, the high susceptibility of commercial chicken lines to histomonosis could be demonstrated and characterized by severe lesions in the ceca and insignificant involvement of the liver, approaching a maximum on days 7-14 postinfection.     

Infection of turkeys with Histomonas meleagridis by the cloacal drop method

The infection of turkeys with Histomonas meleagridis was attempted in the absence of its normal vector Heterakis gallinarum, using several experimental techniques. Battery-reared poults were inoculated at 2 wk of age with histomonads cultured in vitro, by several routes, including (a) per os (PO), (b) intradoacal (CI), and (c) cloacal drop (CD). Feed restriction was also studied as a predisposing factor. Intracloacal inoculation (CI) consistently produced severe infections in all experiments. In several experiments, turkeys did not become infected after inoculation PO with 1 x 10(5) cultured histomonads. Feed restriction prior to inoculation did not make turkeys susceptible to infection inoculated PO. However, when liquid cultures containing histomonads were applied to the vent (CD) and the dorsal lip stimulated to initiate cloacal drinking, the histomonads were taken into the cloaca and transported to the ceca by retrograde peristalsis. Heavy infections were produced by this method, with severe liver and cecal lesions recorded when birds were necropsied 12 days later. These results suggest that CD may provide ready entry into the lower intestinal tract for these parasites and may facilitate spread of infection through flocks.     

Effect of coated plant extracts on Histomonas meleagridis and growth of bacteria in vitro

Three coated plant extracts (RepaXol and two experimental formulations) were tested for their minimal lethal concentration for histomonads in vitro and the effect of those substances on the bacterial growth in the histomonadal culture. After 48 hr, RepaXol and experimental formulation B were lethal to histomonads at a concentration of 1.25 microl/ml. Experimental formulation C was lethal at a concentration of 2.5 microl/ml. All products also decreased the growth of bacteria at concentrations inhibiting the growth of histomonads.     

Detection of Histomonas meleagridis DNA in different organs after natural and experimental infections of meat turkeys

Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.     

Seroprevalence of Histomonas meleagridis in pullets and laying hens determined by ELISA

Serum samples from 1120 layers from 56 flocks and 400 pullets from 20 flocks were tested by an indirect sandwich ELISA to investigate the prevalence of antibodies to Histomonas meleagridis in chickens kept in alternative husbandry systems. The overall prevalence of antibodies to H meleagridis in layers was 37.3 per cent, and positive birds were identified in 50 flocks. This was significantly higher than in pullets, where only 8.3 per cent of the birds tested positive. Optical density (OD) values obtained from pullet sera were much lower than the OD values from layers; however, positive birds were detected in half of the pullet flocks. In particular, all birds from an organic pullet flock were found to be positive, with high OD values. Overall, the highest prevalence of positive sera was obtained from birds kept in free-range flocks. Attempts to reisolate live histomonads from birds in 18 layer flocks were unsuccessful.     

Detection of Histomonas meleagridis in turkeys cecal droppings by PCR amplification of the small subunit ribosomal DNA sequence

Histomonas meleagridis is a protozoan parasite that may cause histomoniasis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis and high mortality. Diagnosis of this disease is based on direct identification or on cultivation of the parasite. With the aim of developing more sensitive, rapid and useful tools for parasite detection, PCR that amplified a DNA target of 209 pb of the 18S rRNA gene was designed to detect the genome of H. meleagridis and to differentiate it from the genome of Tetratrichomonas gallinarum, another common protozoan parasite of fowl. The sensitivity of the test was evaluated using serial diluted samples of cultured H. meleagridis and showed positive amplification for concentrations comprised between 10 and 10(-1)parasites/ml of culture. The sensitivity for cecal droppings samples was assessed using spiked material and was comprised between 3 x 10(3) and 3 x 10(5)parasites/ml of stool. The reliability of the PCR for the detection of Histomonas infection was also evaluated by experimental infection of turkeys. Results of the PCR appeared to be in agreement with the development of the clinical signs and of the cecal lesions. The PCR developed in this study may be a useful tool in the detection and identification of H. meleagridis for rapid, routine screening as a supplement to direct identification or cultivation of the parasite.     

Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the diagnosis of histomoniasis

Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.     

The frequency of detection of Mycoplasma meleagridis in breeding turkeys depending on the laying age

Coating of air sacs was recorded from day-old chicks from parents with Mycoplasma (M.) meleagridis infection, with positive findings being obtained from 9.52% of all animals early in the laying period and from 34.09% up to the 8th laying week. M. meleagridis was isolated from palatine and cloacal swabs taken of laying hens and insemination cocks, with positive findings being 50-60% prior to the laying period (28th week of age), 100% at start of laying, and 80% in the 14th laying week. M. meleagridis was identified in 50% of all embryonated eggs as of the 1st laying week and in 100% as of the 4th week. M. meleagridis was cultured from 30.67% of all sperm samples tested, between the 30th and 46th week of production. Differences were found to exist between individual cocks, with 4 cocks being without M. meleagridis at all. There was usually agreement between positive M. meleagridis findings from sperm and cloacal swabs. M. meleagridis was eliminated from cock sperm by spectinomycin (0.6 mg/ml diluting medium), but M. iowae was not.     

Assessment of the antihistomonal effect of paromomycin and tiamulin

Histomonosis, a parasitic disease of galliformes and sporadically of other birds caused by Histomonas meleagridis, can result in very high mortality, especially in turkeys. The ban on the last antihistomonal drug prompted an urgent search for alternative prevention and treatment strategies. As both paromomycin and tiamulin have been reported to have antihistomonal activity, these antibiotics were investigated in vitro by adding two-fold serial dilutions ranging from 12.5 to 400 microg/mL to cultures of H. meleagridis. Controls (no antibiotics, or 12.5 microg or 400 microg/mL dimetridazole) were included. Parasites were counted after 3, 20, 28, 44, 51, and 71 hours of incubation. Tiamulin did not have a clear antihistomonal effect, but paromomycin had an inhibitory effect at all concentrations tested. The latter antibiotic was subsequently examined in an in vivo study. Five groups of 20 1-day-old poults, matched by weight and sex, were either not treated (infected and uninfected control groups) or treated with paromomycin (100, 200, or 400 ppm) added to their feed. After 2 weeks all groups, except for the uninfected control group, were intracloacally inoculated with 200,000 histomonads per bird. A clear dose-response effect was found for paromomycin. In the 100-ppm paromomycin group, mortality was similar to that in the untreated control group, whereas about half of the birds died in the 200-ppm paromomycin group; almost complete protection against histomonosis was seen in the 400-ppm paromomycin group. This study shows that paromomycin supplied in feed at 400 ppm is a potentially preventive strategy against H. meleagridis.     

Leptin enhances maturation and development of calf oocytes in vitro

We investigated the effects of leptin on the in vitro maturation (IVM) and development of calf oocytes. Cumulus-oocyte complexes were matured in IVM medium containing 0-100 ng/ml leptin. Experiment 1 showed that exposure of calf oocytes to IVM medium containing 1 or 10 ng/ml leptin significantly increased rates of development to the metaphase II stage compared with the control (81.7 ± 3.0% and 83.3 ± 2.1% for 1 and 10 ng/ml leptin, respectively, vs 64.1 ± 5.1% for control; p < 0.05). Experiment 2 showed that 1 or 10 ng/ml leptin significantly improved cleavage rates after in vitro fertilization when compared to control (58.6 ± 3.3% and 59.3 ± 2.9% for 1 and 10 ng/ml leptin, respectively, vs 48.5 ± 2.6% for control; p < 0.05); in addition, when compared to control medium, the addition of 10 ng/ml leptin to the IVM medium resulted in more presumptive zygotes reaching the 4- to 8-cell stage after 48 h of in vitro culture (30.3 ± 2.3% vs 20.1 ± 2.3%; p < 0.05) and developing into blastocysts after 8 days of culture (20.4 ± 1.6% vs 11.7 ± 1.7%; p < 0.05). Experiment 3 showed that the addition of 1 or 10 ng/ml leptin significantly increased the total number of blastocyst cells on day 8 of culture (114.6 ± 7.8 and 117.4 ± 5.9 for 1 and 10 ng/ml leptin, respectively, vs 92.7 ± 8.3 for control; p < 0.05) and trophectoderm (TE) cells (88.5 ± 5.5 and 90.6 ± 3.7 for 1 and 10 ng/ml leptin, respectively, vs 70.1 ± 5.9 for control; p < 0.05). In summary, these results indicate that the addition of leptin to IVM medium enhances meiotic maturation and embryo development from calf oocytes and improves the quality of embryos derived from these oocytes.     

The efficacy of some drugs with known antiprotozoal activity against Histomonas meleagridis in chickens

Nine drugs with known or suspected antiprotozoal activity were tested in vitro, and in vivo for activity against Histomonas meleagridis. The nitroimidazoles dimetridazole, metronidazole, ornidazole, and tinidazole suppressed growth of H. meleagridis in vitro at 10 microg/ml or higher. Paromomycin sulfate, and carbadox were weakly effective at high levels. Quinolinol, mebendazole, diloxanide furoate, and albendazole had no demonstrable efficacy in vitro. Drugs showing some activity in vitro were tested in young chickens inoculated intracloacally with 2 x 10(5) H. meleagridis/bird. Dimetridazole, metronidazole, ornidazole, and tinidazole were highly effective at 200 ppm in feed. Paromomycin sulfate, and carbadox were ineffective in vivo, with no improvement in liver or cecal lesion scores compared to that of infected controls. Thus, the only new entities with efficacy against blackhead disease in vivo were nitroimidazoles, related to the positive control dimetridazole.     

Blackhead disease (Histomonas meleagridis) aggravated in broiler chickens by concurrent infection with cecal coccidiosis (Eimeria tenella).

The effect of concurrent cecal coccidiosis infections on severity of Histomonas meleagridis (blackhead disease) in chickens was investigated in a series of experiments. Cecal lesions from H. meleagridis were severe in all inoculated control groups and did not appear to be affected by the introduction of Eimeria tenella infection. However, the severity of liver lesions and number of birds positive for liver lesions of H. meleagridis increased significantly with the presence of E. tenella. The increase was similar when 10(3) or 10(4) oocysts of E. tenella were given and was the same when oocysts were given at the same time as H. meleagridis or 4 days prior. The liver lesions increased directly as doses of H. meleagridis increased from 7.5 x 10(3) cells to 30, 100, or 300 x 10(3) when E. tenella was given along with H. melelagridis but not when H. meleagridis was given alone. Administration of a live coccidiosis vaccine containing very low levels of E. tenella also gave a significant boost to liver lesions but at a much lower level than that observed with larger doses of E. tenella. The positive relationship between infections of cecal coccidiosis and H. meleagridis in chickens suggests that such dual exposure may contribute to increased clinical outbreaks of blackhead disease in chickens under field conditions.     

Effect of potassium simplex optimization medium and NCSU23 supplemented with beta-mercaptoethanol and amino acids of in vitro fertilized porcine embryos

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4- cell (80.8 to 85.4% vs. 73.6%) and blastocyst (30.4 to 30.5 vs. 23.5%) stages and the number of TE (51.4 to 53.8 vs. 35.8) and total cells (67.2 to 71.2 to 48.8) over the control group. On the other hand, supplementing KSOM with AA and/or beta-ME produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4-cell (78.8% vs. 67.7%) and morula (57.8% vs. 46.3%) stages and the number of ICM (18.6 to 19.2 vs. 11.6) and total cells (62.8 to 70.6 vs. 42.8) over control group. In conclusion, our study demonstrates that both KSOM and NCSU-23 medium supplemented with AA and beta-ME and/or only beta-ME alone are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.     

In Vitro and in Vivo Developmental Competence of Dromedary (Camelus dromedarius) Embryos Produced in Vitro Using Two Culture Systems (mKSOMaa and Oviductal Cells)

Development competence and pregnancy rate of in vitro-produced (IVP) dromedary embryos were studied in two culture systems: (i) semi-defined modified medium (mKSOMaa) and (ii) co-culture using camel epithelial oviducal cells. Five hundred and three cumulus-oocytes complexes (COCs) were selected, allowed to mature, fertilized and cultured in vitro (38.5 degrees C; 5% CO2, maximum humidity > 95%, with concentration of oxygen of 5% for semi-defined medium and 20% for co-culture cells). Maturation was accomplished by incubation in TCM-199 medium supplemented with 10% heat-treated foetal calf serum (FCS), 10 ng/ml epidermal growth factor, 1 microg/ml follicle-stimulating hormone, 1 microg/ml oestradiol and 500 microM cysteamine for 30 h. In vitro fertilization (IVF) was performed using fresh semen (0.5 x 10(6) spermatozoa/ml in modified TALP solution). Fertilized COCs were denuded by vortexing, then cultured in either mKSOMaa (10% heat-treated FCS was added 24 h post-IVF), under 5% O2 and 90% N2 (group 1; n = 249) or with dromedary epithelial oviducal cell monolayers in TCM-199 with 10% heat-treated FCS under 20% O2 (group 2; n = 254). The rate of cleavage was significant higher (p < 0.05) for group 1 (63%, 156/249) than for group 2 (51%, 130/254). No significant difference was found between the two groups in the rate of development to blastocyst (21% vs 16.5%) and their hatchability (21% vs 14%). Pregnancy rates were similar for the first 60 days. However, all pregnancies were lost after 60 days with the exception of two of six (33%) from recipients of hatched blastocysts from group 1. We conclude that both systems support in vitro production of dromedary embryos by in vitro maturation (IVM)/IVF of oocytes. However, embryos obtained by culture in the semi-defined medium (mKSOMaa) appear to have a better in vivo development ability.     

Xylazine-induced hyperglycemia in beef cattle.

Intravenous administration of xylazine to beef cattle (10 animals, 0.2 mg/kg of body weight) resulted in rapid onset (less than 15 minutes) of hyperglycemia. Plasma glucose values increased to 195 +/- 15 mg/dl and 305 +/- 10 mg/dl at 15 minutes and 3 hours, respectively. Concomitantly, plasma insulin concentrations dropped from 23 +/- 2 microU/ml before xylazine to 5.8 +/- 0.7 microU/ml and 2.4 +/- 0.3 microU/ml at 15 minutes and 3 hours, respectively. Parallel decreases (20%) were observed for percentage of hemoglobin, red blood cell number, and packed cell volume. Plasma urea nitrogen was significantly (P less than 0.01) incrased within 3 hours of xylazine administration (6.7 +/- 0.9 mg/dl vs 11.4 +/- 0.7 mg/dl). Marked changes in concentrations of plasma-free fatty acids were not observed. Alternative means of anesthesia must be considered in those instances in which biopsy material is to be used for studies of carbohydrate metabolism in vitro.     

Increased susceptibility of bobwhites (Colinus virginianus) to Histomonas meleagridis after exposure

Bobwhites given heterakid eggs but no Sevin became infected with cecal histomonads, but there was no pathological histomoniasis. Quail given 50 microgram of Sevin (10 microgram/day) behaved normally, but at necropsy they had slightly discolored livers. Quail given various doses of heterakid eggs and Sevin (Sevin increasing from 2.5 to 50 microgram) and those given various doses of heterakid eggs and 10 microgram/day of Sevin developed pathological histomoniasis and mortality rates of 36 and 63%, respectively.     

Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.     

Prediction of rabbit caecal fermentation characteristics from faeces by in vitro gas production technique: roughages

To find equations able to estimate the fermentation characteristics of the caecum from that of faeces, caecal content and faeces of 10 hybrid Hyla rabbits were used as inocula for an in vitro gas production trial. About 1 g of 12 roughages, 11 hays (ryegrass, alfalfa, sulla, oat, vetch, sulla-lolium, vetch-oat, sulla-oat, clover, ryegrass-clover, sulla-vetch-oat) and a wheat straw, was weighed, in triplicate per inoculum, in 120-ml flasks; 75 ml of anaerobic medium and 4 ml of reducing solution were added and the flasks were placed at 39 degrees C. Caecal content and faeces were diluted respectively 1:2 (CI) and 1:8 (FI) with anaerobic medium and were introduced into their respective flasks (10 ml). Gas production was recorded 20 times at 2-24 h intervals throughout fermentation (120 h). The fermentation characteristics (i.e. degraded organic matter, OMd; potential gas production, A; maximum fermentation rate, R(max); volatile fatty acid, VFA; ammonia, NH(3)) were studied by inocula and substrates. The two inocula did not differ in OMd but CI produced significantly higher gas (A, 213.1 vs. 199.4 ml/g, respectively, for CI and FI, p < 0.01) in less time (R(max), 3.08 vs. 2.24 ml/h, respectively, for CI and FI, p < 0.01). CI also produced higher levels of total VFA (57.86 vs. 46.70 mmol/g OM, respectively, for CI and FI, p < 0.01) and showed a higher branched chain proportion (0.023 vs. 0.018, respectively, for CI and FI, p < 0.01). For some parameters (as OMd pH and propionate) the equations for the estimation of caecal fermentation characteristics from that of faeces were accurate (R(2) > 0.8828) and reliable (CV < 10.78%) suggesting that faeces can be successfully used for the estimation of these parameters.     

Performance and oocyst shedding in broiler chickens orally infected with Cryptosporidium baileyi and Cryptosporidium meleagridis

To study effects of experimental cryptosporidiosis, broiler chickens were infected per os with 5 x 10(5) oocysts of Cryptosporidium baileyi and Cryptosporidium meleagridis. In the first experiment, chickens were infected with oocysts of C. baileyi at the age of 7, 14, and 21 days. In the second experiment, chickens were infected with oocysts of C. baileyi, C. meleagridis, or both cryptosporidial species at the age of 7 days. Although clinical signs of infection were apparent, neither final live weight nor mortality was significanty influenced in chickens infected with a single Cryptosporidium species. In chickens infected with C. meleagridis, the growth retardation was observed in the 2-wk period after infection. The compensatory growth, however, started when the oocyst shedding had ceased. The number of oocysts in excreta specimens of chickens infected with C. meleagridis was two to three times lower than in excreta of chickens infected with C. baileyi. Chickens infected with both C. baileyi and C. meleagridis (5 x 10(5) oocysts of each) had significantly lower final live weight and worse feed efficiency than chickens of other groups. Concurrent infection did not influence individual C. baileyi or C. meleagridis oocyst shedding.