Eimeria maxima: characteristics of attenuated lines obtained by selection for precocious development in the chicken

Both the Weybridge strain and a mixture of six laboratory and field strains of Eimeria maxima have been attenuated by selection for early maturation of oocysts during serial passage in chickens. The prepatent times of the resultant precocious lines produces after selection were reduced from approximately 120 hr to less than 107 hr, and their reproduction and pathogenicity were less than those of the parent strains. Chickens which had been inoculated with a small number of oocysts of the Weybridge precocious or mixed field isolate precocious lines were immune to subsequent challenge with the parent strains. In a comparison made between the endogenous development of the precocious lines and their parent strains, it was found that asexual multiplication of the precocious lines was reduced, due to the earlier onset of gametogony. The results of this study also indicate that the parent (i.e., normal) strains of E. maxima probably undergo a minimum of four generations of schizogony.     

Live attenuated mutants of Mycobacterium tuberculosis as candidate vaccines against tuberculosis

The recent advances in genetic tools to manipulate Mycobacterium tuberculosis have led to the construction of defined mutants and to the study of their role in the virulence and pathogenesis of tuberculosis. The safety and vaccine potential of a few of these M. tuberculosis mutants as candidate vaccines against tuberculosis are discussed.     

The endogenous development of virulent strains and attenuated precocious lines of Eimeria tenella and E. necatrix

Studies were made of the endogenous development of the H strains of E. tenella and E. necatrix, and precocious lines derived from them. Second-generation meronts of both precocious lines matured at a faster rate than those of the parent strains. The first- and second-generation meronts of the precocious line of E. necatrix and the second-generation meronts of the precocious line of E. tenella were significantly smaller and contained fewer merozoites than the corresponding stages of the parent strains. These observations provide reasons for the precocity, and a partial explanation for the attenuation, of these lines.     

Eimeria tenella and E. acervulina: lymphokines secreted by an avian T cell lymphoma or by sporozoite-stimulated immune T lymphocytes protect chickens against avian coccidiosis

The role of avian lymphokines as nonspecific immunomodulators of host immunity against the intracellular parasite Eimeria was investigated. Prophylactic treatment of normal chickens with crude cell-free supernatants obtained from JMV-1 culture, concanavalin A (Con A)-stimulated normal spleen cells, or sporozoite-stimulated immune T cells prior to inoculation with E. tenella or E. acervulina conferred significant protection. These crude cell-free culture supernatants also inhibited intracellular development of eimerian parasites in vitro. Avian macrophages pretreated with these supernatant preparations showed inhibitory activity against Eimeria. This inhibitory activity could not be ascribed to anti-Eimeria antibody, complement, or cell-free Marek's disease virus and was therefore considered to be due to immunomodulating lymphokines present in the culture supernatants. These results suggest that JMV-1-transformed T lymphoblastoid cells, immune T lymphocytes, and Con A-stimulated normal spleen cells secrete lymphokines that can enhance host immunity in a nonspecific manner and implicate cell-mediated immunity as a major mechanism of the protective host immune response against eimerian infections.     

Live and killed vaccines against toxoplasmosis in mice

Mice were immunized with live organisms of the different stages (i.e., tachyzoites, bradyzoites, or sporozoites) of Toxoplasma gondii, or with killed tachyzoites with or without adjuvants. The adjuvants used were liposomes, anhydrides of myristic or lauric acid, levamisole and Freund's complete or incomplete adjuvant. The following strains of T. gondii were used: RH, M-7741, the nonpersisting, temperature-sensitive mutants ts-1, ts-4, or ts-5, and the "back mutant" of ts-1 (Pfefferkorn and Pfefferkorn, 1976). The protection afforded was measured by challenge with the pathogenic M-7741 strain. Killed tachyzoites alone, or with adjuvants, offered only slight protection against challenge with M-7741 and no protection against challenge doses that were lethal to all control mice. Chronic infection and live nonpersisting vaccines conveyed a strong immunity to challenge, except strain ts-1. Because it was less pathogenic and did not require chemoprophylaxis, strain ts-4 best fulfilled the requirements for a good vaccine; its effect in hosts other than the mouse remains to be determined. The immunity induced by tachyzoites, bradyzoites, or sporozoites appeared equally strong when challenged with sporozoites.     

Ovine coccidiosis: pathology of Eimeria ovinoidalis infection

Doses of sporulated oocysts of Eimeria ovinoidalis ranging from 10² to 5 × 10⁶ were given to 25 housed lambs aged between 5 and 13 weeks, most of which had been reared coccidia-free. Some had received an “immunizing” dose 3–4 weeks earlier. Lambs were killed between 8 and 21 days after inoculation (DAI) and the tissues were examined histologically. Doses higher than 10⁶ caused extensive loss of epithelial cells in the lower jejunum both from the surface and from the crypts at 10 DAI when first-generation meronts were mature. Doses of 10³ oocysts or more caused diarrhoea from about 13 DAI in both first and second infections; this was associated with massive invasion of the caecal epithelium by second-generation meronts and gamonts. Destruction of crypt stem cells by these stages led to denudation of the caecal mucosa, resulting in haemorrhagic enteritis, dehydration and delayed healing or death.     

Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction (PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of this important disease of poultry.     

Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis

Feline infectious peritonitis (FIP) is a fatal immunity-mediated disease caused by mutants of a ubiquitous coronavirus. Since previous attempts to protect cats under laboratory and field conditions have been largely unsuccessful, we used our recently developed system of reverse genetics (B. J. Haijema, H. Volders, and P. J. M. Rottier, J. Virol. 77:4528-4538, 2003) for the development of a modified live FIP vaccine. With this objective, we deleted the group-specific gene cluster open reading frame 3abc or 7ab and obtained deletion mutant viruses that not only multiplied well in cell culture but also showed an attenuated phenotype in the cat. At doses at which the wild-type virus would be fatal, the mutants with gene deletions did not cause any clinical symptoms. They still induced an immune response, however, as judged from the high levels of virus-neutralizing antibodies. The FIP virus (FIPV) mutant lacking the 3abc cluster and, to a lesser extent, the mutant missing the 7ab cluster, protected cats against a lethal homologous challenge; no protection was obtained with the mutant devoid of both gene clusters. Our studies show that the deletion of group-specific genes from the coronavirus genome results in live attenuated candidate vaccines against FIPV. More generally, our approach may allow the development of vaccines against infections with other pathogenic coronaviruses, including that causing severe acute respiratory syndrome in humans.     

Ovine coccidiosis: the pathology of Eimeria crandallis infection

Doses of sporulated oocysts of Eimeria crandallis ranging from 50 to 300,000,000 were given to 27 housed lambs aged between 4 and 12 weeks that had been reared coccidia-free. Lambs were killed between 1 and 22 days after inoculation and their tissues examined histologically. Clinical effects were very variable and not closely related to inoculating dose. Some lambs showed intermittent diarrhoea, sometimes watery and sometimes containing muco-fibrinous material, either in the form of intestinal casts or as a greyish discoloration. Loss of surface epithelial cells and villus atrophy in the small intestine were associated with first generation meronts and the release of merozoites from them; in some lambs apoptosis of crypt cells also occurred, leading to crypt atrophy. Severe diffuse crypt hyperplasia was associated with pro-gamonts in the small and large intestines. In a minority of the lambs this stage was associated with what appeared to be crypt destruction by host cells in the lamina propria, leading in some cases to denudation and severe diarrhoea.     

Comparative clinical trial of vaccines against avian influenza

Scientic-production association "Microgen" has finished 1st phase of clinical trials of candidate vaccines against avian influenza in order to assess their reactogenicity, safety, and immunogenicity. Two vaccines constructed from NIBRG-14 vaccine strain [A/Vietnam/1 194/2004 (H5N1)], obtained from World Health Organization, were studied: "OrniFlu" (inactivated subunit influenza vaccine adsorbed on aluminium hydroxide) and inactivated polymer-subunit influenza vaccine with polyoxydonium (IPSIV). Clinical trial of the vaccines with different quantity of antigen (15, 30, and 45 mcg of H5N1 virus hemagglutinin) was carried out in Influenza Research Institute (St. Petersburg) and in Mechnikov Research Institute of Vaccines and Sera (Moscow). Analysis of results allowed to conclude that both vaccines were safe, well tolerated and characterized by low reactogenicity. Two-doses vaccination schedule was needed to meet required seroconversion and seroprotection rates (> or =1:40 in > or =70% of vaccinated volunteers). "Orni-Flu" vaccine containing 15 mcg of hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) in one dose as well as IPSIV containing 45 mcg of hemagglutinin and 0.75 mg of polyoxydonium in one dose were most immunogenic after 2 doses - seroprotection rates in microneutralization assay were 72.2% and 77.0% respectively. Marked influence of aluminium hydroxide content on immunogenicity of the "OrniFlu" vaccine was confirmed in the study. Optimal quantity of adjuvant was 0.5 mg per dose. According to basic concept of vaccine development, preference is given to vaccine that under minimal quantity of antigen induces sufficient specific immune response and is safe in volunteers. "OrniFlu" vaccine containing 15 mcg of H5N1 virus hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) corresponded to these requirements that allowed researchers to recommend it for clinical trials of 2nd phase.     

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.     

Differences in Hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella

The levels of expression of Heat shock protein 70 (Hsp 70) in sporozoites of a wild-type parent strain and 2 precocious lines of Eimeria tenella, were compared to investigate the relationship between the heat shock proteins expressed by the parasite and virulence of the strain. Hsp70 expression was analyzed in sporozoites by immunohistochemical techniques, immunoblot, and flow cytometric analyses. One band of 70 kDa was identified and the variation of the Hsp70 expression levels was quantified by optical densitometric analyses. The results showed a significant gradual decrease in the Hsp70 expression in sporozoites of E. tenella as attenuation progressed, suggesting that the Hsp70 expressed in the excysted sporozoites of E. tenella might be involved in parasite pathogenicity. In addition, the cytoplasmic distribution of the Hsp70, which was observed in the entire sporozoites of the wild strain, was reduced to the anterior portion in the precocious lines.     

Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells

Background

Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry.

Results

Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay.

Conclusions

A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.     

Vaccination of mice against murine coccidiosis by ingestion of surface proteins of Eimeria falciformis incorporated in liposomes

Proteins are released from the surface of sporozoites of Eimeria falciformis during their in vitro incubation in a detergent solution. Some of these proteins reacted with antibodies from infected mice and specifically stimulated the proliferation of mesenteric lymph node cells of these mice. Oral immunization of mice with liposome encapsulated sporozoite surface antigens protected mice against a challenge infection. Two proteins (M.W. 27 and 180 K) induced an antibody synthesis in these vaccinated mice.     

Eimeria species and genetic background influence the serum protein profile of broilers with coccidiosis

Background

Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria.

Methodology/Principal Findings

Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001.

Conclusions/Significance

Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.