Baileyi Oocysts Research Articles

Detection of Cryptosporidium baileyi oocysts in the faeces of domesticated fowls in Ninevah governorate

The study was designed to identify Cryptosporidium oocyst with the determination of infection rate in the faeces of local backyard chicken in Ninevah governorate. One hundred and forty freshly voided fecal samples were randomly collected from both sex of local hens aging between 6-12 months old. These samples were taken from ten various regions of the governorate from the 1st Oct. 2010 till 1st Oct. 2011. Two techniques were applied in this study, hot modified acid – fast stain was used to define the parasite species, while iodine stain was followed for determination the infective rates. Using hot modified acid – fast stain, C. baileyi was diagnosed and identified based on the measurements and diameters of the oocyst using ocular micrometer. Such dimensions were (4.6 × 6.2) micron containing four sporozoites. Out of 140 fecal samples, only 59 samples harbored the oocyst parasite in a percentage of 44.5% hens more than 6 month-one year ages and 33.2% for less than 6 months. However, higher infection rates were noticed in the females which were 45% versus 17.9% in the males. The infection rate was variant among different places which ranged between 25% and 55.6 %. There were significant statistical differences (P<0.05) in the infection rates of the geographical regions of the governorate.

Molecular identification and phylogenetic analysis of Cryptosporidium, Hepatozoon and Spirometra in snakes from central China.

Snakes are popular as food and traditional medicine in China. However, information about parasitic and bacterial infections in snakes from China is scarce. We investigated the prevalence of selected zoonotic agents including Cryptosporidium, Hepatozoon and Spirometra, in snakes in central China from June to October in 2018 by PCR amplification using parasite-specific primers. PCR amplification and DNA sequencing showed that 10.1% (15/149) of snakes were positive for Cryptosporidium spp., while 2.7% (4/149) were positive for Hepatozoon. Additionally, we found 36.9% (55/149) of snakes were infected with Spirometra erinaceieuropaei. The spargana burden per infected snake ranged from 1 to 26. BLAST and phylogenetic analysis of small subunit ribosomal RNA (SSU rRNA) gene and 60-kDa glycoprotein (gp60) gene showed that the parasites belonged to Cryptosporidium parvum genotype IIdA15G1, C. baileyi, C. serpentis and a Hepatozoon species. We conclude that intensively farmed snakes excrete C. parvum and C. baileyi oocysts due to ingestion of infected feeder animals, and that wild snakes in central China were commonly infected with S. erinaceieuropaei, suggesting that eating improperly cooked snakes could be risky to human health.

Efficacy of a potential DNA vaccine encoding Cryptosporidium baileyi rhomboid protein against homologous challenge in chickens

The parasite Cryptosporidium baileyi can infect the larynx, trachea, bursa and cloaca of poultry, causing high mortality during severe infection and leading to substantial economic losses of the poultry industry. The rhomboid protein is very important in Cryptosporidium infection. In this study, a nucleic acid based vaccine candidate pEGFP-CbROM was constructed. After orally challenging with C. baileyi oocysts, the corresponding immune responses induced were analyzed and the immunoprotective effect evaluated in chickens. Obtained results revealed that this nucleic acid based vaccine could induce antibody responses and peripheral blood T lymphocytes proliferation significantly (P<0.05), while the peripheral blood B lymphocyte proliferation increased significantly (P<0.05) only at a high dose of 100μg of pEGFP-CbROM, compared with the PBS control group. After C. baileyi infection, the duration of oocysts shedding was shortened by 2days in the 100μg pEGFP-CbROM group, and the rate of reduction could reach to around 71.3%. While no significant difference in body weight gain was observed among the immunized groups (P>0.05), the differences between the immunized and the non-immunized groups were found to be significant (P<0.05). Our data provides a useful basis for further work in cryptosporidiosis prevention and treatment.

An in vitro model of infection of chicken embryos by Cryptosporidium baileyi

Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds. Cryptosporidium baileyi is the most common species reported in a wide range of avian hosts. Although this parasite is well investigated, there is no adequate in vitro model for its endogenous development, and therefore, knowledge of each life cycle phase is scarce. In the present study, an in vitro model for C. baileyi in chicken embryos was developed and the complete life cycle investigated by light and electron microscopy, including both the sexual and asexual reproduction stages. The complete life cycle of C. baileyi was observed during 1–96 h post inoculation (PI), and the average reproduction number of C. baileyi oocysts in allantoic fluid of each chicken embryo was greatest at 168 h PI. These results suggest that chicken embryos could adequately represent the natural host cells and support the development of all the endogenous life cycle stages of C. baileyi, and also provide a new and effective in vitro cultivation system for further studies on antigens, virulence, infectivity, metabolites, and sensitivity of drugs against parasites.

Reduced Serologic Response to Avian Influenza Vaccine in Specific-Pathogen-Free Chicks Inoculated with Cryptosporidium baileyi

Immunosuppression in chickens as a consequence of Cryptosporidium baileyi infection may compromise the effectiveness of vaccination for control of avian influenza (AI) H5N1. In a 13-wk study using 260 specific-pathogen-free chicks, immunological response and bursa weight:body weight ratios following vaccination against AI strain H5N1 were compared between uninfected and C baileyi-infected birds. The four experimental treatments were the following: infection with C. baileyi (5 x 10(5) oocysts dosed orally on day 0); C baileyi infection + vaccination against H5N1 (0.3 ml inactivated vaccine administered subcutaneously on day 7 and day 21; CB+VAC); vaccination only (VAC); and sham infection/vaccinations using sterile distilled water (control). At weekly intervals serum samples were analyzed by hemagglutination inhibition assay (HI), and five chicks/group were euthanatized for determination of bursa weights. In all but week 8, proportional bursa weights (bursa weight/body weight) were lower for CB+VAC chicks than for VAC chicks (i.e., the bursa index was below 1.0; average 0.81). Throughout the 13 wk, HI titres were lower (P < 0.01) in CB+VAC than in the VAC group, and compared with VAC, chicks in CB+VAC had a slightly shorter period of negative seroconversion. These data indicate bursal atrophy and immunosuppressive effects of C. baileyi infection on day-old chicks vaccinated against AI strain H5N1 and suggest that C. baileyi infection in chicks may increase the host susceptibility to AI virus.

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.

Influence of in ovo administered Cryptosporidium baileyi oocyst extract on the course of homologous infection

In order to evaluate the effect of in ovo vaccination on avian cryptosporidiosis, two doses (1 and 10 μg) of Cryptosporidium baileyi oocyst extract (OE) were injected into the amnionic sac of embryonated, specific pathogen-free chicken eggs. After hatching these birds as well as infected controls (IC) were inoculated with 8×10 5 C. baileyi oocysts at 10 days of age. Another group of chickens remained uninfected (UC). Faecal oocyst shedding was measured every second day, and weekly ELISAs were performed to monitor seroconversion. Those chickens that received OE during embryogenesis showed dose-dependent shift in their oocyst shedding, with higher oocyst output of OE1 and OE10 birds compared to IC ones. The patency was significantly longer in the OE10 group than in IC or OE1. ELISA results showed low seroconversion of OE1 and OE10 chickens prior to homologous challenge. Challenge infection resulted in antibody levels without significant difference between IC, OE1 and OE10 groups. These data suggest that in ovo vaccination with C. baileyi oocyst extract does not promote immune response, moreover, it may impair immunity and thus delay the clearance of cryptosporidia from chickens.

Cloning and expression of a DNA sequence encoding a 41-kilodalton Cryptosporidium parvum oocyst wall protein.

This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of oocyst proteins from several different Cryptosporidium species. Antiserum was then prepared against a 41-kDa antigen unique to C. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvum oocysts was confirmed by reverse transcriptase PCR (RT-PCR). Although the CP41 sequence was shown by PCR to be present in the genome of C. baileyi, CP41 mRNA was not detected in this species by RT-PCR. Immunofluorescence staining with antiserum against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy demonstrated that native CP41 was distributed unevenly on the C. parvum oocyst surface and was associated with amorphous oocyst wall material. In an enzyme-linked immunosorbent assay, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological responses of young calves and adult cows to experimental and natural C. parvum infections. These results indicate that recombinant CP41 antigen may have potential in the immunodiagnosis of cryptosporidiosis.

Immunogenicity of three oocyst extracts of Cryptosporidium baileyi in experimentally infected chickens.

The immunogenic properties of water-soluble and detergent-extracted components of Cryptosporidium baileyi oocysts were studied. Oocyst cytosol antigen (OCA) containing hydrophilic proteins was obtained by freeze-thaw cycles in liquid nitrogen. This was followed by Triton X-114 extraction of remaining oocyst fragments to dissolve membrane-bound proteins (TRE). The remainder of the pellet was solubilized with sodium dodecyl sulfate and treated with 2-mercaptoethanol to reduce disulfide-linked oocyst wall proteins (BME). The immune recognition of these three extracts was evaluated during the course of experimental cryptosporidiosis in chickens using ELISA, immunoblotting, and the lymphocyte stimulation test (LST). Four groups of chickens were infected at various times with different doses of C. baileyi and one group with the mammalian parasite C. parvum. Analysis of the data revealed that OCA proteins are well recognized by serum antibodies during the infection and to a limited extent by sera from chickens infected with C. parvum. Humoral responses of chicken groups to this antigen did not correlate well with the length of patency in contrast with its cellular recognition in LST. TRE gave lower values than OCA in both ELISA and LST, though it was still specifically recognized by samples from C. baileyi-infected chickens. Antibodies reacted aspecifically with BME, since only samples of birds which were immunocompetent at the time of their infection were able to recognize this extract as antigen. Immunoblotting revealed more specific components in OCA than in TRE or BME.

Assessment of maternal immunity to Cryptosporidium baileyi in chickens

The degree of protection to Cryptosporidium baileyi in the progeny of infected chickens was studied. Hens at the beginning of their laying period were given orally three consecutive, large doses of C. baileyi oocysts at weekly intervals. The infection became patent after 6 days and lasted for another 6 days. Increasing serum IgG, and serum, bile, lachrymal and salivary IgA were demonstrated from their samples. These immunoglobulins were transferred to the eggs, since high levels of maternally derived IgG and lower amount of IgA were present in their yolks. Hatchlings of infected hens were divided into uninfected (UY) and infected (IY) groups, the birds in the latter receiving an oral inoculum of C. baileyi oocysts on the first day of their life. Two other groups, progeny of uninfected hens served as controls (uninfected UC, and infected IC). Maternal IgG was detected in serum samples of UY hatchlings which was eliminated by the third week. The total oocyst shedding of IY chickens was 54.3% lower than that of the controls (IC), however, the prepatent and patent periods did not show significant difference. In spite of the partial protection observed in IY birds, their humoral immune response to C. baileyi was significantly lower when compared to IC. A dot-ELISA was developed to evaluate seroconversion of infected chickens which was 100% in both infected groups. The findings of the present study suggest that infection of hens with C. baileyi results in partial protection of their progeny to this parasite, and factors other than immunoglobulins may also be transferred via the eggs.

Interaction of chicken anaemia virus and Cryptosporidium baileyi in experimentally infected chickens

The natural occurrence of concomitant chicken anaemia virus (CAV) and Cryptosporidium baileyi infection was described earlier. In this experiment, 1-day-old chickens were infected with CAV alone (anaemia virus infected, AI) or followed by inoculation with 8×10 5 C. baileyi oocysts orally at 1 wk of age (anaemia virus and Cryptosporidium infected, ACI). Another group of chickens received the same dose of C. baileyi oocysts without previous virus infection ( Cryptosporidium infected, CI), and two groups of uninfected chickens served as controls. Except one group (uninfected control, UC), all groups — including the other control group (challenged control, CC) — were challenged with an oral inoculum of 8×10 5 C. baileyi oocysts at the age of 4 wk. Haematological, serological, immunohistochemical and pathological findings confirmed the effect of the virus agent. The individual C. baileyi oocyst shedding did not show significant difference between group CI and ACI, however, after challenge infection the AI chickens shed approximately three times more C. baileyi oocysts than those in group CC. Mortality and the percentage of birds that developed anaemia was significantly higher among ACI than AI chickens, while haematocrit values at 2 wk of age and relative bursal weights at 4 wk of age were moderately lower in the ACI group. The results presented here suggest that concurrent CAV infection increases the reproductive potential of C. baileyi in chickens, and both pathogens have synergistic effect on each other.

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187–1.5 mg kg −1 and T-2 toxin levels of 0.187–6.0 mg kg −1 were investigated. The experimental amimals were orally infected with 6 × 10 5 C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0–6.0 mg kg −1) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg −1 T-2 toxin in the feed significantly depressed body weight gain and immunity.

Effects of Cryptosporidium baileyi infection on the bursa of Fabricius in chickens.

In order to clarify the effect of cryptosporidiosis on immune response, histopathological changes associated with experimentally occurring bursal cryptosporidiosis in chickens were chronologically observed as the first step. A total of 150 2-day-old chickens was each inoculated orally with a single dose of 5 x 10(5) Cryptosporidium baileyi oocysts. The chickens showed a normal profile of oocyst shedding in droppings. The bursa indices throughout the experimental period indicated negligible reactions. Numerous cryptosporidia occurred in the microvillous border of bursal epithelium between days 4 and 16 postinoculation (PI). Appearance of the most mast cells was followed by a dramatic loss of the protozoa in the bursa of Fabricius (BF). The distribution of the coccidium coincided with heterophil infiltration in the epithelium and adjacent lamina propria. The histopathological lesion was marked diffuse chronic superficial purulent bursitis with heterophil infiltration in the epithelium and adjacent lamina propria and mucosal epithelial hyperplasia. These results suggest that the bursitis may induce immunosuppressive effect.

Attempts to Immunize Chickens with Cryptosporidium baileyi Oocyst Extract

In order to study the possibility of immunization against Cryptosporidium baileyi with extracted crude antigen, Arbor Acres chickens were injected intramuscularly with 80 micrograms of C. baileyi oocyst-derived proteins (uninfected immunized, UI) or inoculated orally with 8 x 10(5) viable C. baileyi oocysts (infected control, IC) at 1 wk of age. The immunization was repeated in the UI group at 2 wk of age. Uninfected (UC) birds served as controls. All animals in UI, IC, and UC groups were challenged orally with 8 x 10(5) C. baileyi oocysts at the age of 4 wk. Blood samples were collected when birds were 4 and 6 wk of age, and sera were examined by enzyme-linked immunosorbent assay for the presence of antibodies against C. baileyi. Total oocyst output of UI chickens was about 60% of that of UC birds after challenge, and the prepatent and patent periods were nearly identical in the latter 2 groups. In contrast, IC birds developed complete resistance to challenge infections. These results suggest that immunization with the oocyst extract of C. baileyi may confer some degree of protection against oral challenge; however, the protection is less effective than that induced by primary oral infection. The lack of significant difference between the antibody responses of IC and UI animals to C. baileyi at 2 wk of age suggests that serum antibodies play little role in acquired resistance to challenge infection.

Kinetics of serum antibody responses in broiler chicks againstCryptosporidium baileyi

The kinetics of serum antibody responses in broiler chickens against Cryptosporidium baileyi were studied. Broilers were inoculated intratracheally with 250,000 C. baileyi oocysts at 1, 7, or 14 days of age. Antibody was quantified by an enzyme linked immunosorbent assay. Anti-cryptosporidial serum immunoglobulins (IgM and IgG) were detected 9 days post-inoculation (DPI) in birds inoculated at 1 or 7 days of age with oocysts and 4 DPI when 14-day-old birds were inoculated. Results also reaffirmed age related susceptibility, with day-old birds being more susceptible than 7-day, and 14-day-old birds were not susceptible to clinical disease. The susceptibility to infection correlated with the amount and duration of the IgM response. Day-old inoculated birds developed a higher, longer-lasting response than 7 or 14-day-old infected birds.

Experimental Biliary Cryptosporidiosis in Broiler Chickens

Biliary cryptosporidiosis was studied by inoculation of 2 x 10(5) Cryptosporidium baileyi oocysts (AU-B1 isolate) into the gall bladders of ten 6-day-old broiler chickens. Clinical signs of disease were not seen. Three of the 10 chickens developed biliary tract infections, based on histologic examination of tissue sections. Lesions seen in the gall bladders of these birds included epithelial hyperplasia and infiltration of the underlying connective tissue with mononuclear leukocytes. One of these birds also had involvement of the hepatic bile ducts. The bile ducts were mildly dilated and contained lesions similar to those seen in the gall bladder. Few to many cryptosporidia were present in the gall bladders and bile ducts of infected birds. Chickens may be of use in the study of biliary cryptosporidiosis, a common sequel to enteric infection in humans with human immunodeficiency virus infection.

Experimental Infections in Domestic Ducks with Cryptosporidium baileyi Isolated from Chickens

Oral inoculation of 13 ducks (Anas platyrhynchos) with 1 x 10(6) Cryptosporidium baileyi oocysts produced patent infections but no clinical signs of disease. Intratracheal inoculation of 13 ducks with 1 x 10(6) C. baileyi oocysts produced only mild clinical signs of respiratory disease, no deaths, and gross lesions of airsacculitis in only three ducks. The distribution of developmental stages of C. baileyi in ducks was similar to that observed in experimentally infected chickens and turkeys. Results of this study indicate that ducks are more resistant to experimentally induced respiratory cryptosporidiosis caused by C. baileyi than are chickens and turkeys.

Experimental Cryptosporidiosis and Infectious Bursal Disease Virus Infection of Specific-Pathogen-Free Chickens

Specific-pathogen-free chickens orally inoculated at 4 days of age with a moderately pathogenic vaccine strain of infectious bursal disease virus (IBDV) and/or at 5 days of age with Cryptosporidium baileyi oocysts remained free of overt clinical signs throughout a 16-day period postinoculation (PI). The prepatency period for C. baileyi oocyst shedding was shorter in chickens receiving higher numbers of oocysts, but once shedding was detected, there were no obvious differences in shedding patterns among groups receiving 10(3) through 10(6) oocysts. On days 8 and 16 PI, cryptosporidia were located primarily in the bursae of Fabricius. IBDV exposure was associated with bursal follicle atrophy, whereas C. baileyi infection resulted in bursal epithelial hypertrophy and hyperplasia, mild follicle atrophy, and heterophil infiltration of the bursal mucosa. Examination of experimental groups of 30 birds each indicated that concurrent infection with both agents resulted in more severe bursal lesions, more infected birds, and greater numbers of cryptosporidia in infected tissues. At the termination of the trial, 16 days PI, Cryptosporidium infection was associated with a 6% decrease in mean body weight compared with controls.

Development of and Serologic Evaluation of Acquired Immunity to Cryptosporidium baileyi by Broiler Chickens

Eighteen 2-wk-old broiler chickens that were inoculated orally with oocysts of Cryptosporidium baileyi (Group B) became infected, shed large numbers of oocysts in their feces on Days 6 to 12 post inoculation (PI), and suddenly cleared the parasites from the mucosal epithelium of the bursa of Fabricius (BF), cloaca, or both BF and cloaca on Days 14 to 16 PI. Eighteen uninoculated birds (Group A) did not shed oocysts during this time period. Five days after all birds in Group B stopped shedding oocysts, the 36 chickens comprising both groups were challenged orally with C. baileyi oocysts. At the time of necropsy, 10 days after oral challenge, all Group A birds (previously uninfected, challenged) had heavy C. baileyi infections in the mucosal epithelium of the BF or cloaca. No parasites were found in the BF or cloaca of the Group B birds (previously infected, recovered, challenged) at the time of necropsy.These data demonstrate that a single intestinal infection with C. baileyi can elicit an immune response of sufficient magnitude to clear the parasite from the intestinal (BF and cloaca) mucosa and to make broiler chickens resistant to subsequent oral challenge with oocysts of the same species. Development of resistance to reinfection was accompanied by the appearance of serum antibodies to C. baileyi that were detectable by two Cryptosporidium- specific assays; an indirect immunofluorescent antibody assay and an enzyme-linked immunosorbent antibody assay (ELISA). The ELISA described herein can now be incorporated into serologically based health monitoring programs.

Experimental Cryptosporidiosis in Broiler Chickens

Seven-day-old conventional broilers were inoculated either orally or intratracheally (IT) with 2.5 × 105, 5.0× 105, or 2.0 × 106 oocysts of Cryptosporidium baileyi (32 birds for each dosage level per group; 192 birds total). Thirty-two birds served as unninoculated controls. Mean weekly weight gain and feed conversion were determined during a 5-week period. Carcass pigment was graded using a Roche Color Fan®. Fecal oocysts were calculated from random cage samples 6, 8, 11, 13, 15, 18, 20, 22, and 25 days after inoculation (DAI). Effects of C. baileyi on immune responses were examined for Newcastle disease virus-hemagglutination inhibition (NDV-HI) antibody, infectious bursal disease virus-enzyme-linked immunosorbent assay (IBDV-ELISA) antibody titers and delayed hypersensitivity (DH) in half of the birds in each group.Disease or death from cryptosporidiosis did not result from oral inoculation of C. baileyi. Signs of respiratory disease, consisting of rales, sneezing, and dyspnea were observed in all IT-inoculated birds 7 to 21 DAI. Seven deaths occurred in the IT-inoculated groups 14 to 21 DAI. At necropsy, lung parenchyma was gray, firm, and wet in the ventral region. Air sacs contained a foamy, white to gray, mucoid fluid. Histologic lesions in the air sacs and bronchi were epithelial hyperplasia, discharge of mucocellular exudate to the mucosal surface, thickening of the mucosa by cellular infiltrates, loss of cilia, and dilation of mucous glands. Weight gains for IT-inoculated birds were lower (P<.05) than controls from 14 to 21 DAI, although weight gains for the 5-week period were not significantly different. Pigmentation scores for birds given C. baileyi orally at medium and high levels and for all IT-inoculated birds were significantly lower than controls. Oocyst output was similar regardless of route or level of inoculation. No significant differences were noted in IBDV-ELISA titers between control and C. baileyi-infected chicks; however, significantly increased NDV-HI titers and decreased DH indices were observed in chicks that received C. baileyi oocysts IT at 5.0 × 105 and 2.0 × 106.