In vitro excystation of Cryptosporidium baileyi from chickens

Release of sporozoites from the oocysts of Cryptosporidium baileyi is described from Nomarski interference-contrast microscopy. Just prior to excystation, the four sporozoites became motile and rearranged themselves within the oocyst. The sporozoites were then rapidly expelled through an opening that formed in the oocyst wall, and the residuum was either released or retained within the oocyst. Excysted sporozoites were crescent shaped and measured 5.0-9.0 microns X 1.0-1.6 micron (mean = 6.8 X 1.1 microns). Excystation occurred when sodium taurocholate or a mixture of trypsin and sodium taurocholate was present in the incubation medium. High levels of excystation occurred at 37 degrees or 40 degrees C, but excystation did not occur at 4 degrees C. The ability of biles from two avian and two mammalian hosts to produce excystation of C. baileyi was also studied. After a 2-h incubation at 40 degrees C, the percentages of excystation were 69.5% in goat bile, 45.0% in pig bile, 33.0% in chicken bile, and 34.5% in turkey bile.     

In Vitro Excystation of Spironucleus muris

ABSTRACT. In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia , were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.     

Viability of preserved Cryptosporidium baileyi oocysts

The present study was undertaken to determine the viability and infectivity of oocysts of Cryptosporidium baileyi that had been stored from 1 to 40 months at 4 degrees C preserved in 2.5% potassium dichromate solution. Oocysts of C. baileyi were purified from the feces of experimentally infected chickens using discontinuous sucrose gradients. Subsequently, the purified oocysts were suspended in 2.5% potassium dichromate solution at a concentration of 1 x 10(7) organism/ml, and their viabilities were assessed by nucleic acid staining, histologic examination, and infectivity to 2-day-old chickens. All chickens inoculated with oocysts that had been stored for 1-18 months developed patent infections, while chickens infected with older oocysts remained uninfected. Between 5.8% and 82.2% of the oocysts, stored at 4 degrees C in 2.5% potassium dichromate solution, were found to be viable, as determined by nucleic acid staining. Parasite colonization in the bursa of Fabricius was detected in the microvillus border of bursal epithelium. The finding that C. baileyi oocysts remain infective to chickens for at least 18 months offers important time-saving advantages to investigators who frequently require large numbers of oocysts.     

贝氏隐孢子虫在鸡体内发育的形态学特征及其对寄生器官的影响

取 2日龄海兰雏鸡 5 0只 ,分为 5组 ,分别接种 0、 0 8× 10 6、 1 6× 10 6、 3 2× 10 6、 6 4× 10 6个鸭源贝氏隐孢子虫 (Cryptosporidiumbaileyi)卵囊。接种后在不同的时间间隔内剖杀雏鸡 ,取法氏囊、气管和喉头。扫描电镜观察发现贝氏隐孢子虫主要寄生在鸡的喉头、法氏囊、气管。裂殖体有 2种类型 :Ⅰ型裂殖体含 8个裂殖子 ,Ⅱ型裂殖体含 4个裂殖子。子孢子或裂殖子在钻入过程中 ,虫体逐渐由香蕉形过渡到鼓槌形 ,最后形成球形的滋养体。带虫空泡分为有球形残体的带虫空泡和无球形残体的带虫空泡。观察到小配子的释放和虫体寄生于杯状细胞的现象。贝氏隐孢子虫寄生于气管引起纤毛倒伏、排列紊乱、纤毛融合、脱落 ;致使法氏囊上皮肿胀 ,法氏囊粘膜表面形成皱褶 ,微绒毛脱落、融合、排列紊乱 ,粘液性分泌物增多 ,炎性细胞渗出     

Factors Affecting Motility and Morphology of Cryptosporidium Sporozoites In Vitro1

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH < 6.2) triggering the lysis.     

The Life Cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) Infecting Chickens

ABSTRACT. The life cycle and morphology of a previously undescribed species of Cryptosporidium isolated from commercial broiler chickens is described. The prepatent period for Cryptosporidium baileyi n. sp. was three days post oral inoculation (PI) of oocysts, and the patent period was days 4–24 PI for chickens inoculated at two days of age and days 4–14 for chickens inoculated at one and six months of age. During the first three days PI, most developmental stages of C. baileyi were found in the microvillous region of enterocytes of the ileum and large intestine. By day 4 PI, most parasites occurred in enterocytes of the cloaca and bursa of Fabricius (BF). Mature Type I meronts with eight merozoites first appeared 12 h PI and measured 5.0 × 4.9 μm. Mature Type II meronts with four merozoites and a large granular residuum first appeared 48 h PI and measured 5.1 × 5.1 μm. Type I meronts with eight short merozoites and a large homogeneous residuum first appeared 72 h PI and measured 5.2 × 5.1 μm. Microgamonts (4.0 × 4.0 μm) produced 16 micro-gametes that penetrated into macrogametes (4.7 × 4.7 μm). Macrogametes gave rise to two types of oocysts that sporulated within the host cells. Most were thick-walled oocysts (6.3 × 5.2 μm), the resistant forms that passed unaltered in the feces. Some were thin-walled oocysts whose wall (membrane) readily ruptured upon release from the host cell. Sporozoites from thin-walled oocysts were observed penetrating enterocytes in mucosal smears. The presence of thin-walled, autoinfective oocysts and the recycling of Type I meronts may explain why chickens develop heavy intestinal infections lasting up to 21 days. Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Cryptosporidium baileyi did not produce infections in suckling mice and goats or in two-dayold or two-week-old quail. One of six 10-day-old turkeys had small numbers of asexual stages only in the BF. Four of six one-day-old turkeys developed mild infections only in the BF, and sexual stages of the parasite were observed in only one of the four. All seven one-day-old ducks and seven two-day-old geese developed heavy infections only in the BF with all known developmental stages present.     

The Effects of Reducing Conditions, Medium, pH, Temperature, and Time on in Vitro Excystation of Cryptosporidium

ABSTRACT. Whereas excystation of sporozoites from oocysts of most coccidian species requires exposure to reducing conditions followed by pancreatic enzymes and bile salts, sporozoites of a bovine isolate of Cryptosporidium excysted without exposure to either reducing conditions or to pancreatic enzymes and bile salts. Without prior exposure to reducing conditions, a high percent excysted after incubation in a mixture of trypsin and bile salts in Ringer's solution; fewer excysted after incubation in tap water, even fewer after incubation in salt solutions, and none after incubation in saliva. Excystation, generally greater at pH 7.6 than at pH 6.0 and at 37°C than at 20°C, was observed as early as 1 h after incubation in water or the trypsin-bile mixture. These findings provide circumstantial evidence that oocysts of Cryptosporidium can excyst in extraintestinal sites and liberate sporozoites that can initiate autoinfection.     

Isolation and identification of Cryptosporidium from various animals in Korea. III. Identification of Cryptosporidium baileyi from Korean chicken]

Each of SPF chicken (Hi-Line strain, 2-day-old males) was inoculated with 2.5 or 5 x 10(4) oocysts by stomach tube. The oocyst was the medium type of Cryptosporidium previously isolated from Korean chicken origin, and passed in 2-day-old SPF chicken. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of the developmental stages, the bursa of Fabricius of the chicken was examined by transmission electron microscopy (TEM) on the 12th day postinoculation. The prepatent period for 8 chicken was 5.9 days postinoculation on the average, and the patent period was 12.9 days. The number of oocysts discharged per day for the chicken was reached peak on day 12 postinoculation on the average. A large number of oocysts was found in fecal samples obtained from inoculated chicken on days 8-14 postinoculation. The ultrastructural feature of almost every developmental stage of the medium type from chicken was very similar to that of Cryptosporidium previously isolated from mammalia including human and birds except for the attachment site of C. muris to the mucus cell from mammalia, but dimension of the oocysts from fecal samples of the medium type was different from those of C. meleagridis and mammalia origin. The above results reveal that the medium type of Cryptosporidium of Korean chicken origin is identified as Cryptosporidium baileyi.     

Oxygen Consumption of Coccidial Sporozoites During in Vitro Excystation

SYNOPSIS. Oxygen consumption of sporozoites of Eimeria acervulina, E. necatrix , and E. meleagrimitis was determined during in vitro excystation. Increase in O₂ consumption occurred only when the permeability of the oocyst membrane was altered by either grinding or incubation in cystine buffer with CO₂ when excystation fluid was present. Addition of 10⁻³ M KCN completely inhibited respiration. The O₂ consumption reached a peak during the time the sporozoites were excysting, and then decreased to a lower level where it remained steady while the sporozoites were awaiting stimulus to penetrate host cells.     

Quantification of In Vitro and In Vivo Cryptosporidium parvum Infection by Using Real-Time PCR

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.     

A chicken embryo model for the maintenance and amplification of Cryptosporidium parvum and Cryptosporidium baileyi oocysts

Cryptosporidium is a genus of apicomplexan parasites that inhabit the respiratory and gastrointestinal tracts of vertebrates. Research of these parasites is limited by a lack of model hosts. This study aimed to determine the extent to which infection at the embryo stage can enhance the propagation of Cryptosporidium oocysts in chickens. Nine-day-old chicken embryos and one-day-old chickens were experimentally infected with different doses of Cryptosporidium baileyi and Cryptosporidium parvum oocysts. Post hatching, all chickens had demonstrable infections, and the infection dose had no effect on the course of infection. Chickens infected as embryos shed oocysts immediately after hatching and shed significantly more oocysts over the course of the infection than chickens infected as one-day-olds. In chickens infected as embryos, C. baileyi was found in all organs except the brain whereas, C. parvum was only found in the gastrointestinal tract and trachea. In chickens infected as one-day-olds, C. baileyi was only found in the gastrointestinal tract and trachea. Chickens infected as embryos with C. baileyi died within 16 days of hatching. All other chickens cleared the infection. Infection of chickens as embryos could be used as an effective and simple model for the propagation of C. baileyi and C. parvum.     

Polyphasic typing of Cryptosporidium baileyi: a suggested model for characterization of cryptosporidia

The present study was undertaken to characterize the oocyst morphology, host specificity, organ location, virulence, and sequences of the small subunit ribosomal RNA, 70-kDa heat shock protein, and oocyst wall protein genes of Cryptosporidium baileyi, and to compare this strain with other Cryptosporidium species. This study also aims to serve as a model for polyphasic (phenetic and genetic) characterization of Cryptosporidium species and strains. On the basis of these results, further genetic and phenetic characterization of an avian isolate is needed if the difference between the length or width, or both, of oocysts of an isolate and of C. baileyi is > or = 10% or if the difference between the oocyst shape index of the isolate and of C. baileyi is > or = 3% (or both). The isolate is infectious for mammals or lower vertebrates, or the host range is narrow, i.e., infectious only for some bird species; after oral or intratracheal inoculation, the parasites are not located in the cloaca and in the bursa of Fabricius or the respiratory tract; clinical disease or weight gain reduction can be observed after oral inoculation; the genetic distance for the examined gene between C. baileyi and the isolate is similar in magnitude to that observed between most closely related Cryptosporidium species.     

Immunosuppressive effect of Cryptosporidium baileyi infection on vaccination against avian infectious bronchitis in chicks

Two-day-old commercial chicks were inoculated orally with 2 × 10⁶ oocysts of Cryptosporidium baileyi and vaccinated with 10^3.5 EID₅₀/head of a commercially available avian infectious bronchitis (IB) live virus vaccine at 4 and 14 days following inoculation. Chicks infected with C. baileyi were shown to have an immunosuppressive effect on IB virus. It is concluded that infection with the protozoon in early life may increase their susceptibility to IB.     

In Vitro Activity of Coumermycin A1

The in vitro activity of coumermycin A(1) was compared with that of novobiocin, ampicillin, and minocycline. Coumermycin was found to be the most active antibiotic of the four against Staphylococcus aureus. It was about 50 times more active than novobiocin or minocycline against the strains tested. Coumermycin also showed good activity against group A streptococci and pneumococci, moderate activity against Escherichia coli, indole-positive Proteus species, and Pseudomonas aeruginosa, and poor activity against Klebsiella-Enterobacter and enterococci. Against P. mirabilis, however, coumermycin activity was almost equal to that of ampicillin. The new antibiotic was further found to be greatly reduced in activity in the presence of plasma, but its minimal inhibitory concentration was not greatly affected by inoculum size. Coumermycin was found to be bacteriostatic in its action, and resistance to it developed slowly. Also, cross-resistance was present with novobiocin but absent with ampicillin or minocycline.