Study on the course of Cryptosporidium baileyi infection in chickens treated with interleukin-1 or indomethacin.

The effects exerted by human recombinant interleukin-1 beta (hrIL-1 beta) and the prostaglandin inhibitor indomethacin on the course of Cryptosporidium baileyi infection in chickens were studied. Daily oocyst shedding was monitored by a quantitative method throughout the experiment. Humoral immune response to C. baileyi was assessed by ELISA at 3 weeks of age while the level of cellular immune response to phytohaemagglutinin-P (PHA-P) by a skin test at 23 days of age. Parenteral application of hrIL-1 beta decreased oocyst shedding to 62%, but the infection ran a similar course in treated and control birds. The PHA-P skin test demonstrated increased cellular immune reaction in chickens receiving IL-1 beta, but there was no significant difference in the humoral responses of the two groups as detected by ELISA. On the other hand, indomethacin mixed to the feed lessened oocyst shedding to 13.7% and also shortened its duration. Immunological parameters as reflected by PHA-P skin test and ELISA results indicated enhanced cellular but unaltered humoral immune response. These data suggest that the systemic application of interleukin-1 can induce partial protection against C. baileyi in chickens and that prolonged, abundant oocyst shedding is due to an indomethacin-sensitive immunodepression via the prostaglandin pathway.     

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.     

2株贝氏隐孢子虫鸡源分离株对雏鸡的致病性比较

为了解不同地区鸡源贝氏隐孢子虫的致病特点,对收集到的郑州、林州两地区鸡源贝氏隐孢子虫卵囊经雏鸡传代扩增纯化后,分别以1×106个卵囊量接种3日龄罗曼公雏鸡,从其排卵囊情况、临床症状和病理学变化比较了2个分离株的致病情况。结果表明:2个隐孢子虫分离株均主要引起雏鸡呼吸道症状和法氏囊炎病变;接种雏鸡均于感染后第4天开始排卵囊,林州株和郑州株排卵囊持续期分别为23 d和13 d;排卵囊高峰期均为感染后第8~12天。雏鸡感染2个地区鸡源贝氏隐孢子虫分离株后,排卵囊量及排卵囊规律存在差异。     

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.     

鸭源贝氏隐孢子虫对鸡的致病性

设计3个不同剂量的鸭源隐孢子虫试验感染2日龄雏鸡,通过临床症状、增重、排卵囊规律和法氏囊指数及寄生器官组织扫描电镜观察,证实贝氏隐孢子虫160万个卵囊感染即可引起明显的呼吸道症状,发病鸡增重显著降低,法氏囊严重萎缩。贝氏隐孢子虫致病程度和排卵囊规律与感染剂量相关,主要病变表现呼吸道病变和法氏囊炎症。     

Interaction of Marek's disease virus and Cryptosporidium baileyi in experimentally infected chickens

Histocompatible B13/B13 white specific-pathogen-free leghorn chickens were used to investigate the effect of coinfection with Cryptosporidium baileyi and the HPRS 16 strain of Marek's disease virus (MDV) in chickens and to assess the pathogenicity of C. baileyi when MDV is given before or after the parasite. Groups of chickens concurrently infected with C. baileyi orally inoculated at day (D)4 and MDV inoculated at hatching (C4M0 group) or at D8 (C4M8 group) were compared with relevant control groups inoculated with only C. baileyi at D4 (C4 group), only MDV at hatching (M0 group) or at D8 (M8 group), and an uninoculated control group (UC group). The chickens were kept in isolator units until the end of the experiment at D62. Our results showed a considerable synergistic effect in concurrently infected chickens and more severe consequences when chickens received MDV before C. baileyi infection. In fact, except for a slight transitory weakness, the chickens in C4 group remained free of overt clinical signs and there was no mortality. However, coinfection with both pathogens induced more lasting or permanent oocyst shedding. Severe clinical cryptosporidiosis with weakness, anorexia, depression, growth retardation, and chronic and severe respiratory disease causing death occurred in all chickens in the C4M0 group between D12 and D43 and in 67% of the chickens in the C4M8 group between D17 and D57. Eighty-two percent and 33%, respectively, died before the development of specific Marek's disease lesions. Mortality rates were 27% and 33% in the M0 and M8 groups, respectively. The presence of MDV enhanced the establishment of more lasting cryptosporidial infection in the respiratory tract, esophagus, crop, proventriculus, and kidneys (only in C4M0 group) as well as in bursa of Fabricius, ceca, and cloaca. Serologic analysis showed that chickens with chronic cryptosporidiosis in the C4M8 group had an increased level of C. baileyi-specific immunoglobulin A. Our results may explain some cases of mortality in chickens naturally infected with MDV and Cryptosporidium.     

固始鸡源隐孢子虫人工感染试验

为了解来源于地方鸡品种的隐孢子虫分离株致病特点,对收集到的河南固始鸡源隐孢子虫经鹌鹑传代纯化后,进行动物感染。结果:固始鸡源隐孢子虫分离株无论在正常还是免疫抑制情况下均不能感染小鼠,但能成功感染海兰雏鸡,出现明显的呼吸道症状及法氏囊病变。剖检发现虫体主要寄生在法氏囊、气管和泄殖腔等部位。根据卵囊形态学及寄生部位等特点,本试验分离的隐孢子虫种类鉴定为贝氏隐孢子虫(Crypto-sporidium baileyi)。增大感染剂量,可使雏鸡排卵囊高峰期提前,排卵囊量增大,持续期延长;免疫抑制剂的使用也可使高峰期提前,持续期延长,但会造成试验动物死亡率增高。雏鸡临床症状、剖检病变和增重减少均与感染剂量呈正相关,免疫抑制剂的使用会加重此影响。     

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.     

Immune complex vaccines for chicken infectious anemia virus

Infection of maternal, antibody-negative chickens with chicken infectious anemia virus (CIAV) can cause clinical disease, while infection after maternal antibodies wane often results in subclinical infection and immunosuppression. Currently, vaccines are not available for vaccination in ovo or in newly hatched chickens. Development of CIAV vaccines for in ovo use depends on the ability to generate vaccines that do not cause lesions in newly hatched chicks and that can induce an immune response regardless of maternal immunity. Immune complex (IC) vaccines have been successfully used for control of infectious bursal disease, and we used a similar approach to determine if an IC vaccine is feasible for CIAV. Immune complexes were prepared that consisted of 0.1 ml containing 10(5.4) tissue culture infective dose 50% of CIA-1 and 0.1 ml containing 10 to 160 neutralizing units (IC Positive [ICP]10 to ICP160), in which one neutralizing unit is the reciprocal of the serum dilution required to protect 50% of CU147 cells from the cytopathic effects caused by CIA-1. Virus replication was delayed comparing ICP80 and ICP160 with combinations using negative serum (IC Negative [ICN]80 or ICN160). In addition, the number of birds with hematocrit values <28% were decreased with ICP80 or ICP160 compared to ICN80 or ICN160. Seroconversion was delayed in ICP80 and ICP160 groups. To determine if ICP80 or ICN 160 protected against challenge, we vaccinated maternal, antibody-free birds at 1 day of age and challenged at 2 wk or 3 wk of age with the 01-4201 strain. Both ICP80 and ICP160 protected against replication of the challenge virus, which was measured using differential quantitative PCR with primers distinguishing between the two isolates. Thus, in principle, immune complex vaccines may offer a method to protect newly hatched chicks against challenge with field virus. However, additional studies using maternal, antibody-positive chicks in combination with in ovo vaccination will be needed to determine if immune complex vaccines will be useful to protect commercial chickens.     

不同禽源贝氏隐孢子虫分离株对鹌鹑致病性的比较研究

本研究旨在观察不同禽源贝氏隐孢子虫(Cryptosporidium baileyi)对鹌鹑的致病性差异。采用光镜、扫描电镜等技术研究了鸡源、鸭源、鹌鹑源、白文鸟源、鸵鸟源贝氏隐孢子虫分离株对10日龄鹌鹑的致病性。结果表明,感染鹌鹑均出现腹泻和呼吸道症状,排卵囊持续期11~18 d,虫体寄生于喉头、气管、法氏囊和泄殖腔。其中鸭源C.baileyi感染组鹌鹑发病率和死亡率分别为82.5%、25%,鸡源C.baileyi(郑州株)感染组发病率和死亡率分别为77.5%、12.5%,鹌鹑源C.baileyi感染组发病率、死亡率分别为37.5%和17.5%。本研究结果提示,不同禽源C.baileyi对鹌鹑有一定致病性,其致病性强弱存在差异。     

Coccidiosis immunization: effects of mushroom and herb polysaccharides on immune responses of chickens infected with Eimeria tenella

An experiment was conducted to investigate the effects of polysaccharide extracts (E) of two mushrooms, Lentinus edodes (LenE) and Tremella fuciformis (TreE), and an herb, Astragalus membranaceus (AstE), on the immune responses of chickens infected with Eimeria tenella. A total of 180 broiler chickens were assigned to nine groups: three groups were fed with each of the extracts (LenE, TreE, and AstE), three groups were fed with the extracts and immunized with live oocyst vaccine (LenE+V, TreE+V, and AstE+V), a group was immunized with the vaccine only, and there were two controls (E. tenella-infected and noninfected groups). The oocyst vaccine was given at 4 days of age, and the extracts (1 g/kg of the diet) were supplemented from 8 to 14 days of age. At 18 days of age, all birds except those in the noninfected group were infected with 9 x 10(4) sporulated oocysts. The results showed that at 7 days postinfection (p.i.), birds fed the extracts without vaccination had lower body weight (BW) gain than those given the vaccine only. However, the extracts in conjunction with the vaccine significantly enhanced BW gain of the infected chickens compared with the vaccine group. Of the three extracts, LenE and TreE showed a better growth-promoting effect. The extracts largely increased oocyst excretion of droppings during the primary response postvaccination. The cecal peak oocyst output and lesion scores measured at 7 days p.i. were higher in the groups fed the extracts than in the group immunized with the vaccine only, whereas those of the groups fed with the extracts and immunized with the vaccine were not significantly different from the vaccine group. Of the three extracts, both LenE- and AstE-fed groups showed lower cecal oocyst output. Thus, as compared with the extracts, the live, attenuated vaccine showed better results with significantly increased immune response in coccidial infected birds. The polysaccharide extracts may prove useful against avian coccidiosis, and, particularly when they are used in conjunction with vaccine, they have shown preliminary promise against the experimental coccidial infection.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

Interaction of reovirus and Cryptosporidium baileyi in experimentally infected chickens

No clinical signs, gross lesions, or increased mortality were observed in specific-pathogen-free chickens orally inoculated at 5 days of age with Cryptosporidium baileyi, reovirus 2035, reovirus 2408, or combinations of these agents. Weight gain of chickens inoculated with only reovirus 2408 was depressed 0-8 days postinoculation (PI) (P less than 0.01) but not for the 21-day period PI. Weight gain of chickens inoculated with only reovirus 2035 was not affected. Cryptosporidium baileyi infection significantly depressed weight gain 8-14 days PI but not for the entire 21-day period PI. Weight gain of chickens infected with both C. baileyi and reovirus 2035 was significantly depressed 0-14 days PI and for the entire 21-day period PI. Dual infection with C. baileyi and either reovirus appeared to promote shedding of both agents. Cryptosporidia were found principally in the rectum 2-10 days PI and in the bursa of Fabricius 6-10 days PI. Reovirus infection did not cause any microscopic lesions and did not modify lesions caused by C. baileyi infection.     

Prevalence of Cryptosporidium baileyi in ostriches (Struthio camelus) in Zhengzhou, China

Few data are available on the molecular characterization of Cryptosporidium spp. in ostriches. The objective of this study was to determine the prevalence of Cryptosporidium species or genotypes in ostriches. A total of 452 fecal samples from five farms, a zoo, and an animal rescue center in Zhengzhou, Henan Province, China were examined for Cryptosporidium oocysts by microscopy of wet mount of fecal materials concentrated by the Sheather's sugar flotation technique. Fifty-three samples were Cryptosporidium-positive from four farms, with an overall prevalence of 11.7%. The percentage of animals shedding oocysts was 0, 16.2%, 7.2%, and 0 in 1-3 weeks, 4-8 weeks, 3-12 months, and more than 12 months ostriches, respectively (χ(2)=17.74; ρ<0.01). PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene of the 53 Cryptosporidium-positive samples showed the presence of only Cryptosporidium baileyi, which was confirmed by DNA sequencing of the SSU rRNA PCR products from 16 positive samples. Cross-transmission studies demonstrated that the C. baileyi isolate could infect chickens and quails. Thus, ostriches are commonly infected with C. baileyi that is genetically and biologically similar to C. baileyi found in other birds.     

Avian influenza in ovo vaccination with replication defective recombinant adenovirus in chickens: vaccine potency, antibody persistence, and maternal antibody transfer

Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibody persistence, transfer of maternal antibodies (MtAb), and interference between MtAb and active in ovo or mucosal immunization with RCA-free recombinant Ad expressing a codon-optimized AIV H5 HA gene from A/turkey/WI/68 (AdTW68.H5(ck)). Vaccine coverage and intrapotency test repeatability were based on anti-H5 hemagglutination inhibition (HI) antibody levels detected in in ovo vaccinated chickens. Even though egg inoculation of each replicate was performed by individuals with varying expertise and with different vaccine batches, the average vaccine coverage of three replicates was 85%. The intrapotency test repeatability, which considers both positive as well as negative values, varied between 0.69 and 0.71, indicating effective vaccination. Highly pathogenic (HP) AIV challenge of chicken groups vaccinated with increasing vaccine doses showed 90% protection in chickens receiving > or = 10(8) ifu (infectious units)/bird. The protective dose 50% (PD50) was determined to be 10(6.5) ifu. Even vaccinated chickens that did not develop detectable antibody levels were effectively protected against HP AIV challenge. This result is consistent with previous findings ofAd-vector eliciting T lymphocyte responses. Higher vaccine doses significantly reduced viral shedding as determined by AIV RNA concentration in oropharyngeal swabs. Assessment of antibody persistence showed that antibody levels of in ovo immunized chickens continued to increase until 12 wk and started to decline after 18 wk of age. Intramuscular (IM) booster vaccination with the same vaccine at 16 wk of age significantly increased the antibody responses in breeder hens, and these responses were maintained at high levels throughout the experimental period (34 wk of age). AdTW68.H5(ch)-immunized breeder hens effectively transferred MtAb to progeny chickens. The level of MtAb in the progenies was consistent with the levels detected in the breeders, i.e., intramuscularly boosted breeders transferred higher concentrations of antibodies to the offspring. Maternal antibodies declined with time in the progenies and achieved marginal levels by 34 days of age. Chickens with high maternal antibody levels that were vaccinated either in ovo or via mucosal routes (ocular or spray) did not seroconvert. In contrast, chickens without MtAb successfully developed specific antibody levels after either in ovo or mucosal vaccination. These results indicate that high levels of MtAb interfered with active Ad-vectored vaccination.     

EFFECT OF ENVIRONMENTAL TEMPERATURE ON SUSCEPTIBILITY OF YOUNG CHICKENS TO SALMONELLA TYPHIMURIUM

Day-old chickens kept in a cold environment (18 degrees to 22 degrees C) were more susceptible to a low and moderate challenge of Salmonella typhimurium than chickens similarly challenged and kept in a warm environment (32 degrees to 36 degrees C). Cold stress at 10 degrees C for 24 h when applied to 12-day-old chickens effectively increased the number of birds shedding organisms. However a similar cold stress on 20-day-old chickens resulted in a less dramatic increase in the number of birds shedding organisms. Of the 60 birds previously challenged with S. typhimurium and then subjected to cold stress, 16 birds recommenced shedding and 7 birds with no previous history of shedding began to shed organisms.     

Comparison of in ovo and post-hatch vaccination with particular reference to infectious bursal disease. A review

In ovo vaccination is an alternative approach to post-hatch vaccination of chickens, particularly in broilers. Vaccination at embryonation day 18 helps to 'close the window' of susceptibility i.e. the time between vaccination and early exposure to infectious agents compared with post-hatch vaccination. Attempts on embryonal vaccination as a mode of vaccine delivery were approached from the observation that chickens already develop certain immunologic functions before hatching. The immune system in birds begins to develop early during embryogenesis and various immune reactions have been induced in the late stage chicken embryos. Compared with post-hatch vaccination, in ovo vaccination stimulates both the innate and adaptive immune responses with the advantage that because of the prenatal immunization, in ovo vaccinated chicks have developed an appreciable degree of protection by the time of hatch. Effects of maternal antibodies on vaccines to be used for in ovo vaccination can be prevented by developing vaccines that are insensitive to maternal antibodies. It has been described that vaccination of chicken embryos at embryonation day 18 did not significantly affect the immune competence of hatched chickens. The apparent absence of tolerance in chicks hatched from embryos exposed to an antigen at the late stage of embryonation implies the feasibility of in ovo vaccination. Investigations on in ovo vaccination to produce safe and efficient vaccines are still in progress. Currently a large number of vaccines are under investigation for viral, bacterial and protozoal diseases.     

Susceptibility and protection of naive and vaccinated racing pigeons (Columbia livia) against exotic Newcastle disease virus from the California 2002-2003 outbreak

The susceptibility, immune response, and protection to challenge after vaccination in racing pigeons (Columbia livia) was assessed with the 2002-2003 exotic Newcastle disease (END) virus responsible for the most recent major outbreak in Southern California. Immunologically na?ve pigeons appeared resistant to disease, regardless of dose, after a natural route of exposure. Twenty percent morbidity was observed in each group of birds receiving between 10(2.1) and 10(8.1) 50% embryo infectious dose (EID50) per bird, with one bird succumbing to challenge in the 10(8.1) EID50/bird group at day 12 postinoculation. Although resistant to disease, birds in all groups continued to shed virus from either oral or cloacal route at the end of the 14-day sampling period, and seroconversion was only observed in birds receiving > or =10(6.1) EID50. Single or double vaccination of juvenile and adult birds with pigeon paramyxovirus virus type 1 (PPMV-1) vaccine followed by END challenge with 10(6.1) EID50/bird decreased the duration, incidence, and viral load. A positive correlation was observed between the presence of hemagglutination-inhibiting antibody titers at challenge and decreased viral shedding. Overt clinical signs of disease were not observed in any PPMV-1-vaccinated birds after challenge.