The correlation of protective effects and antibody production in immunized chickens with recombinant R7 vaccine against Leucocytozoon caulleryi

Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (> or = 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (< or = 3,200) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis.     

Field efficacy of recombinant R7 vaccine against chicken leucocytozoonosis

Effectiveness of vaccine that used recombinant R7 protein (rR7) as antigen that is derived from second-generation schizont (2GS) of Leucocytozoon caulleryi was verified under a field condition against chicken leucocytozoonosis. Chickens reared in a poultry farm where the chickens are attacked by leucocytozoonosis in every year were inoculated with oil-adjuvanted rR7 vaccine (O-rR7), and the immunized chickens were found to have production of antibodies against 2GS at a high level by one shot. Leucocytozoonosis was observed at post-injection. During the epidemic period of leucocytozoonosis, the unique clinical signs of the disease such as discharge of green feces and anemia, and also parasitemia were observed, however, compared to chickens in control group, those in O-rR7 vaccinated group had significantly slight symptoms (P<0.05). In addition to this, immunized chickens had better result of egg production than the unvaccinated chickens did, and the maximum difference of egg production rate, 22%, was observed at the peak of the disease. In conclusion, it is verified that O-rR7 vaccine has efficacy against leucocytozoonosis under field condition, and this vaccine can be put into practical use.     

Culicoides arakawae (Diptera: Ceratopogonidae) population succession in relation to leucocytozoonosis prevalence on a chicken farm in Taiwan

Leucocytozoonosis caused by Leucocytozoon caulleryi is a significant disease prevalent in open chicken houses of southern and eastern Asia. L. caulleryi is transmitted by Culicoides arakawae, a blood-sucking vector. Leucocytozoonosis prevalence is influenced by vector population succession. Thus this examination was performed on a farm to investigate vector population succession and leucocytozoonosis prevalence in experimental chicks and to obtain the ecology data for assessing the prevalence. The findings were as follows: (1) C. arakawae adults might be highly host specific because they were rarely discovered on cattle or pig farms, and none of the experimental chickens were infected by L. caulleryi on those farms. (2) Identifying and counting gorged and gravid C. arakawae to assess leucocytozoonosis prevalence is a practical strategy. The critical vector index should be 5.0 calculated by dividing the smallest vector mean from the prevalent period by the largest vector mean from the population not causing leucotocytozoonosis. (3) Taking vector means from three or more collections each month, should be the best assessment of leucocytozoonosis prevalence because C. arakawae succession appears to have a 3-week periodicity. Hopefully, these findings will contribute to assessing leucocytozoonosis prevalence.     

The extraintestinal stages of Eimeria tenella and E. maxima in the chicken

Donor chickens given feed medicated with one or two levels of decoquinate or given non-medicated feed were infected with oocysts of Eimeria tenella or E. maxima per os. Twelve hours after inoculation with oocysts liver, mid-intestine or ceca homogenates were fed to previously uninfected recipient chickens. The results showed that continuous medication with decoquinate was effective in preventing the transfer of sporozoites from the intestine to the liver. Oocysts were detected in the feces of all recipients of tissue from non-medicated donors, showing that some sporozoites of E. maxima and E. tenella are normally transferred to liver. Young broiler chickens were immunized by oral inoculation of E. maxima oocysts. The immune status of similar chickens inoculated with sporozoites of the same species directly into the liver or spleen were assessed. During the experimental period half of the chicks were provided with non-medicated food and the remainder were given feed supplemented with decoquinate; decoquinate was effective in arresting the development of the sporozoites. Two weeks after initial infection the birds were challenged with oocysts of E. maxima per os. Injection of sporozoites into the spleen did not protect against challenge. Birds inoculated with sporozoites into the liver were unable to develop a significant level of immunity. When the drug pressure was removed from these birds, parasitism of the intestine occurred and immunity developed.     

Demonstration of circulatinf antibodies to Eimeria tenella by the indirect immunofluorescent antibody test using sprozoites and second-stage schizonts as antigen

In the indirect immunofluorescent antibody (IFA) test using sporozoites as an antigen, sera from chickens immunized via the natural route were positive in dilutions as high as 1 : 1 048. Serum from a rabbit, immunized only to sporozoites by subcuntaneous injection, was positive in a dilution of 1 : 4 4 096. Non-immunized chicken andn rabbit sera were positive in dilutions varying from 1 : 20 to 1 : 64.Sporozoites within sporocytes were not stained. In frozen sections of infected chorioallantoic membranes and ceca, sporozoites were not traced with the IFA test with immunized chicken or rabbit sera. However, with the rabbit serum specific diffuse intraepithelial and subepithelial fluorescence was observed in the ceca from 4 to 11 h after infection.Fluorescence was never associated with the first-stage schzonts and gamates, but second-stage schizonts were positive with both chicken and rabbit serum. The titres obtained with this antigen were about the same as those obtained with sporozoite smears. The possible presence of common antigens in sporozoites and second stage schizints is discussed.     

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.     

Characterization of monoclonal antibodies against the second-generation schizonts of Leucocytozoon caulleryi

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.     

Increase of antibody titer against Leucocytozoon caulleryi by oral administration of recombinant R7 antigen

In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

Activation of macrophages by culture fluid of antigen-stimulated spleen cells collected from chickens immunized with Eimeria tenella

The effect of spleen cell factors on the activation of macrophages was investigated in chickens immunized with Eimeria tenella. The abilities of peritoneal macrophages obtained from normal chickens to kill sporozoites of E. tenella and to inhibit intracellular development of Toxoplasma gondii were enhanced by exposing them to 33 and 50% culture fluid of antigen-stimulated spleen cells of chickens immunized with E. tenella. The parasiticidal activity of normal macrophages was also distinctly enhanced by the treatment with culture fluid of phytohemagglutinin-stimulated normal spleen cells. On the other hand, the parasiticidal activity of normal macrophages could not be enhanced by the treatment with culture fluid of antigen-stimulated normal spleen cells under conditions similar to those of culture fluid of antigen-stimulated immune spleen cells. It thus appears that the macrophage activator was induced from immune spleen cells in response to the stimulation by the antigen.     

Experimental transmission of Cytauxzoon felis from bobcats (Lynx rufus) to domestic cats (Felis domesticus)

Freshly collected blood and/or spleen homogenate from an experimentally infected Florida bobcat (Lynx rufus floridanus), which had died of feline cytauxzoonosis, was inoculated into domestic cats. All inoculated cats had clinical signs of feline cytauxzoonosis and died within 2 weeks after they were inoculated. Similar material collected from an eastern bobcat (Lynx rufus rufus) carrying an experimentally infected Cytauxzoon felis parasitemia was inoculated into domestic cats. All inoculated cats developed a parasitemia, but none developed clinical signs of disease and none died of the disease. Cats subinoculated with parasitemic cat blood also developed parasitemias and they too did not develop clinical signs of infection nor died. After carrying the blood phase of Cytauxzoon felis for various periods, the domestic cats were then challenge exposed with proven lethal Cytauxzoon inoculum of domestic cat origin. All cats died of cytauxzoonosis.     

Embryo vaccination of specific-pathogen-free chickens with infectious bursal disease virus: tissue distribution of the vaccine virus and protection of hatched chickens against disease

Vaccination of specific-pathogen-free chickens as 18-day embryos with the BVM isolate of infectious bursal disease virus (IBDV) resulted in extensive replication of the vaccine virus in the embryonic tissues. The virus was recovered from lung, thymus, proventriculus, liver, kidney, and spleen of embryos 1 day postvaccination, and recoverable virus persisted for at least 7 days. Replication and spread of the vaccine virus in chickens vaccinated as 18-day embryos was compared with that in chickens vaccinated at hatch. Distribution of the virus in tissues was more extensive, virus levels in tissues were generally higher, and detectable virus persisted longer in chickens vaccinated as 18-day embryos than in those vaccinated at hatch. Effective vaccine response could be initiated with 6.2 median embryo lethal doses, the lowest dose tested. Chickens immunized as embryos developed neutralizing antibody against IBDV and resisted challenge with pathogenic IBDV at 4, 6, 8, and 10 weeks of age.     

Avian eimeria: invasion in foreign host birds and generation of partial immunity against coccidiosis

Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.     

Cellular immune response in the small intestine of two broiler chicken lines orally inoculated with malabsorption syndrome homogenates

We studied the cellular immune response against malabsorption syndrome (MAS) in two broiler chicken lines, A and B. We determined the number of pan T-lymphocytes (CD3), helper T-lymphocytes (CD4), cytotoxic T-lymphocytes (CD8) and macrophages/monocytes in the small intestine in the first 2 weeks after oral inoculation of two MAS homogenates, MAS80 and MAS97-1. The immune cells were detected on cryostat tissue by immunohistochemistry and counted by villus area. In trial 1, we compared the two broiler lines for weight gain depression, intestinal lesion and number of CD3, CD4, CD8 cells and macrophages/monocytes after MAS80 inoculation. Although there was no significant difference in weight gain depression between the two broiler lines, line B had significantly higher numbers of CD8+ T-cells per villus area than had line A. To confirm part of the results of trial 1, trial 2 was done in which we compared different homogenates in broiler line B. Broiler line B was orally inoculated with either MAS97-1, intestinal homogenate obtained from healthy chickens (healthy homogenate), or phosphate buffered saline (PBS). In this trial, the MAS97-1 homogenate also induced weight gain depression and intestinal lesions, whereas the "healthy homogenate" and PBS did not induce weight gain depression or intestinal lesions. The broilers inoculated with MAS97-1 homogenate had significantly more CD8+ T-cells per villus area than had broilers inoculated with "healthy homogenate" or PBS. Increased CD8+ T-cells per villus area in the affected small intestines of broilers suggests an increase of cytotoxic T-cell activity.     

Adjuvant effect of chicken interferon-gamma for inactivated Salmonella Enteritidis antigen

The adjuvant effect of chicken interferon-gamma (ChIFN-gamma) was examined for protecting chickens against intestinal colonization of Salmonella Enteritidis (SE) following oral exposure. Ten 7-week-old chickens per group were immunized with inactivated SE twice with or without co-administration of ChIFN-gamma intramuscularly, and all chickens were challenged with SE. Sera collected from immunized groups with or without ChIFN-gamma, and from unimmunized group were measured for SE antibody by agglutination test. The levels of antibodies were raised by 1 week post-immunization and did not show any difference between groups with and without ChIFN-gamma. No antibodies were detected in unimmunized group before challenge. Fecal samples from each group were cultured at 1, 4, 7, and 13 days post-challenge to determine the incidence of intestinal colonization and the numbers of SE shed into the environment. Co-administration of ChIFN-gamma, significantly reduced the incidence of intestinal colonization (P<0.05). At 13 days post-challenge, the bacterial counts of SE in organs were also reduced in ChIFN-gamma administered group. These data suggest co-administration of ChIFN-gamma with SE antigen enhances protection against SE challenge without acceleration of antibody production.     

CpG寡核苷酸对IBDV VP2基因真核表达质粒免疫增效作用

以传染性法氏囊病病毒(IBDV)VP2蛋白基因表达质粒DNA为免疫原，以CpG的寡核苷酸(CpG-0DN)为免疫佐剂，肌肉注射于14日龄SPF鸡，1周后加强免疫1次，2次免疫后15d和21d分别测定血清ELISA抗体效价，并于免疫后21d用IBDV99儿强毒株攻毒和进行病理学观察。结果显示，(1)VP2基因重组质粒DNA与CpG共同免疫组的ELISA抗体水平明显高于VP2重组质粒免疫组；(2)IBD弱毒苗与VP2重组质粒免疫组抗体水平明显高于VP2重组质粒免疫组，且比VP2基因重组质粒DNA与CpG共同免疫组略高；(3)VP2基因重组质粒DNA与CpG共同免疫组及IBD弱毒苗与VP2重组质粒免疫组可明显降低IBDV强毒攻击后引起的急性发病率和死亡率。由此表明，CpG寡核苷酸对IBDV VP2蛋白基因真核表达质粒免疫具有明显增强作用，有很大的应用前景。     

鸡脑脊髓炎的病理学诊断

某养殖户送来10只疑似脑脊髓炎的17日龄病鸡。经询问此鸡群共5100只，2日龄开始发病，2~3日龄少数死亡，6~7日龄死亡增多，每天死亡20只左右，到12日龄死亡更多，每天死亡100只左右。临床症状表现为：共济失调，头颈震颤，头上下扭曲，打转。为了进一步确诊和提供更有效的防治方法，笔者对该病例鸡进行了剖检，并采取病料，采用血清学、病理学等诊断方法。结合发病日龄、临床症状、病理变化、ELISA检测等，综合诊断为脑脊髓炎。     

In situ production of interferon in tissues of chickens exposed as embryos to turkey herpesvirus and Marek's disease virus

Chicken eggs at embryonation day (ED) 18 or newly hatched chicks were inoculated with turkey herpesvirus (HVT), Marek's disease virus (MDV), or virus-free diluent and, at intervals after inoculation, tissue homogenates of virus-exposed and virus-free chickens or chicken embryos were examined for interferon (IFN) activity. Homogenates of lung, thymus and spleen specimens from chickens given HVT at ED 18 had IFN activity. Activity of IFN in the lungs was studied further. Homogenates of lung specimens from chickens exposed to HVT at hatching also had IFN activity, although the concentration of IFN was lower than that in chickens given HVT at ED 18. The pathogenic isolates of MDV (JM-MDV), but not the attenuated (Md11/75C-MDV) or nonpathogenic (SB1-MDV) isolates, inoculated at ED 18 also induced high lung IFN activity. Exposure to a combination of HVT and SB1-MDV induced IFN activity comparable with that in chickens given HVT alone. The IFN activity in homogenates of lung specimens from virus-exposed chickens was species specific and heat and pH stable, but was destroyed by trypsin treatment. Occasionally, low IFN activity also was detected in homogenates of tissue specimens from virus-free chickens or chicken embryos. This IFN activity could have been produced constitutively or may have been induced by substances (inducers) in the environment.     

Preparation and evaluation of chicken embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens

The current study was planned to develop an efficient vaccine against hydropericardium syndrome virus (HSV). Currently, formalin-inactivated liver organ vaccines failed to protect the Pakistan broiler industry from this destructive disease of economic importance. A field isolate of the pathogenic hydropericardium syndrome virus was adapted to chicken embryos after four blind passages. The chicken embryo-adapted virus was further serially passaged (12 times) to get complete attenuation. Groups of broiler chickens free from maternal antibodies against HSV at the age of 14 days were immunized either with 16th passage attenuated HSV vaccine or commercially formalized liver organ vaccine. The antibody response, measured by enzyme-linked immunosorbent assay was significantly higher (P < 0.05) in the group immunized with the 16th passage attenuated HSV vaccine compared to the group immunized with liver organ vaccine at 7, 14, and 21 days post-immunization. At 24 days of age, the broiler chickens in each group were challenged with 10^3.83 embryo infectious dose₅₀ of pathogenic HSV and were observed for 7 days post-challenge. Vaccination with the 16th passage attenuated HSV gave 94.73% protection as validated on the basis of clinical signs (5.26%), gross lesions in the liver and heart (5.26%), histopathological lesions in the liver (1.5 ± 0.20), and mortality (5.26%). The birds inoculated with liver organ vaccine showed significantly low (p < 0.05; 55%) protection estimated on the basis of clinical signs (40%), gross lesions in the liver and heart (45%), histopathological lesions in the liver (2.7 ± 0.72), and mortality (35%). Birds in the unvaccinated control group showed high morbidity (84%), mortality (70%), gross (85%), and histopathological lesions (3.79 ± 0.14) with only 10% protection. In conclusion, this newly developed HSV vaccine proved to be immunogenic and has potential for controlling HSV infections in chickens.     

鸡传染性贫血免疫对肝微粒体抗氧化酶活性的影响

目的是观察鸡传染性贫血多次免疫对肝微粒体中抗氧化酶活性的影响.对20只SPF鸡随机分为2组,每组10只,免疫组鸡用鸡传染性贫血弱毒苗免疫4次,每次间隔2周,对照组鸡注射同剂量的生理盐水.最后一次免疫后10d取肝脏制备微粒体,利用测试盒测定肝微粒体中的GSH-Px活性、SOD活性、CAT活性和MDA含量.结果与对照组相比,免疫组肝微粒体中GSH Px活性、SOD活性和CAT活性都显著提高(P＜0.05),MDA含量显著减低(P＜0.05).结论为鸡传染性贫血多次免疫可提高鸡体抗氧化能力.