Regulatory requirements for vaccine authorisation.

Vaccines are one of the most important tools available in the prevention and control of diseases in animals. It is therefore of the utmost importance that when vaccines are used, such use should meet with the requirements of the World Organisation for Animal Health Terrestrial Animal Health Code and must be authorised by the recognised licensing body in the country/region where the vaccines are to be used, in accordance with the three key criteria of quality, safety and efficacy. This article provides a comprehensive and comparative description of the regulatory requirements in place for veterinary vaccines in major regions of the world, highlighting the similarities and pointing out also where there are differences. Recent advances in harmonisation of such testing requirements achieved through the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) are also described. The contents will provide a valuable guide to those engaged in the research and development of vaccines globally, and reassure those involved in the prevention and control of animal diseases that veterinary vaccines, when fully authorised and used according to the label instructions, are safe and efficacious.

Regulations and procedures in parasite vaccine development.

Although immunisation protocols for a wide variety of parasitic diseases have been developed, it is often questioned why these do not always reach the market. In this review information about the regulations and procedures that apply to licensing the production and marketing of medicinal preparations, especially parasite vaccines, is presented. These general regulations specify issues on product (quality, safety, efficacy and potency) and production (facilities and consistency). Vaccine developers and manufacturers have to comply with these regulations, which may involve years of research and development. Moreover, where the manufacturer claims specific features of the product, these claims have to be corroborated by (experimental) data. A series of principles has been used to develop vaccines against parasite infections varying from the use of (attenuated) live vaccines to killed vaccines and subunit vaccines. The implications of some specific regulatory issues associated with these approaches are discussed.

Comparison of 16 chelonid herpesviruses by virus neutralization tests and restriction endonuclease digestion of viral DNA.

A total of 16 chelonid herpesviruses that were isolated between 1992 and 1998 were compared with one another on the basis of serology and restriction enzyme digestion patterns of viral DNA. The viruses stem from tortoises of three different species in four different European countries and the United States of America. The majority of the isolates were similar to one another. One isolate, however, differed strongly from all others both serologically and in the restriction cleavage pattern of its DNA, showing that there are at least two different sero- and genotypes of herpesviruscs that infect tortoises.

Differentiation of psittacine herpesvirus field isolates by restriction endonuclease analysis.

Thirty-one viruses were isolated from 15 different psittacine species. They were all identified as herpesviruses on the basis of chloroform sensitivity, inhibition of replication in cell cultures by 5-iodo-2'-deoxyuridine and morphology by electron microscopy. DNA from all 31 field isolates of psittacine herpesviruses (PsHV) was analysed by cleavage with restriction endonucleases EcoRI, PstI, and BglII. Using this technique, 12 different restriction patterns were recognizable. In previous work, we have differentiated PsHV strains according to their serological interrelationships. Restriction endonuclease profiles confirmed the results of conventional serogrouping but additional differences were observed among the five established serological subtypes. Using this method, it was possible to demonstrate a spontaneous reinfection of parrots in an aviary with a serologically and genetically different PsHV strain 4 years after the first outbreak of Pacheco's parrot disease (PPD). The results of this study reaffirm that antigenetically and genetically different PsHV exist and can cause PPD.

Paramyxoviral and reoviral infections of iguanas on Honduran Islands.

Thirty-five free-ranging healthy spiny-tailed iguanas (31 Ctenosaura bakeri, 4 C. similis) and 14 green iguanas (Iguana iguana rhinolopha) were caught and held in captivity for 2 days. Blood was collected from all animals and their sera were evaluated for antibody titres against reptilian reoviruses, reptilian paramyxoviruses, and avian paramyxovirus-1 (PMV-1). Cloacal and pharyngeal swabs also were collected and examined for viral content by incubation on chicken embryo fibroblasts (CEF) and terrapene heart cells (TH-1). No virus was isolated from the pharyngeal and cloacal swabs on CEF and TH-1. Twenty-three (47%) of 49 sera samples tested positive for reptilian reoviruses by virus neutralization tests. Twenty (41%) of 49 samples had antibodies against one reptilian PMV isolate by virus neutralization tests and 3 (9%) of 34 by hemagglutination inhibition tests. No antibodies were detected against the other PMV isolate of reptilian origin nor against avian PMV-1. This is the first description of serum antibodies against reptilian reoviruses and PMV in wild iguanas.

Quantification of the herpesvirus content in various tissues and organs, and associated post mortem lesions of psittacine birds which died during an epornithic of pacheco's parrot disease (PPD).

This paper reports on the viral content of up to 52 tissue and organ samples of 18 individual large psittacines which died during an epornithic of Pacheco's parrot disease (PPD) caused by psittacid herpesvirus 1 (PsHV1). Associated clinical signs and pathological lesions are described. The large spectrum of samples found to be positive for PsHVl suggests that birds succumb to PPD during viraemia. Tissues and organs from which the virus could be isolated included the integument and associated structures, the muscular, respiratory and circulatory system, bone marrow, the nervous system, thyroid and adrenal glands, spleen and liver, the urogenital tract and the gastro-intestinal tract. Nevertheless, individual and organ (but not species)-specific variation does occur. Virus isolation appears to be most promising from the respiratory, vascular and nervous system and the liver. Highest titres were obtained from heart blood and liver (up to 7.6 log(10)/g tissue), airsac, Nervus vagus and pulp and quill of pin feathers. Pin feathers may therefore be suitable for in-vivo diagnosis. In contrast, HV could not be isolated from any of the feather vanes examined. For the most part, post mortem lesions do not reflect the organ pattern found to be most permissive for virus replication as judged by the success of virus isolation and virus titres. A closer quantitative correlation is indicated for the lungs, spleen and liver, only. Corresponding findings as to frequency of gross pathological lesions and virus quantification appear to be restricted to the liver. In accordance with clinical observations and experimental findings, tissue virus content indicates that horizontal spread of herpesviruses is mediated by cloacal contents or secretions from the respiratory system.

Psittacine beak and feather disease infected cells show a pattern of apoptosis in psittacine skin.

Skin biopsies from 23 birds with psittacine beak and feather disease (PBFD) were examined by light and electron microscopy. Affected cells, preferentially found in the cell layers of the feather follicles, could be clearly identified by the presence of intracytoplasmic virus inclusion bodies. Ultrastructurally, the degenerative process in these cells was morphologically suggestive of apoptosis.

Herpesviruses in tortoises: investigations into virus isolation and the treatment of viral stomatitis in Testudo hermanni and T. graeca.

Various studies were done during a spontaneous outbreak of stomatitis-rhinitis-complex (mouth rot) in a collection of Mediterranean land tortoises (21 Testudo hermanni, Hermann's tortoises, and three Testudo graeca, spur-thighed tortoises) in southern Germany. These studies were intended to help diagnose the causative agent, establish a possible diagnostic method in vivo and provide information on the efficacy of aciclovir and ganciclovir against chelonian herpesviruses. Thirteen T. hermanni and no T. graeca died within a period of 6 weeks following the introduction of one apparently healthy T. graeca. Two of the dead Testudo hermanni were submitted for post-mortem examination. In addition, blood samples from 11 of the 12 tortoises still surviving at the beginning of this study were cultured for virus content and for the presence of neutralizing antibodies to chelonian herpesviruses and swabs from conjunctiva, pharynx and cloaca were cultured for the presence of viruses. Herpesviruses were isolated from tissues of the two dead Testudo hermanni (tongue, intestine, trachea, lung, spleen, heart and brain). Peripheral leukocytes from one of 11 blood samples were positive for herpesvirus isolation, indicating viremia in at least one animal. Nine of 11 pharyngeal swabs but none of the conjunctival and cloacal swabs yielded herpesviruses. Circulating neutralizing antibodies were present in two of two tested T. graeca, but absent in all of the nine samples from T. hermanni. Aciclovir and ganciclovir were effective when tested in vitro against one of the herpesvirus isolates.

Comparison of the genomes of 15 avian herpes-virus isolates by restriction endonuclease analysis.

Differentiation of herpesvirus isolates has been performed mostly on the basis of biological properties and serology. In this study, 15 herpesvirus isolates from different species of birds were compared by restriction endonuclease analysis of genomic DNA. All herpesviruses were isolated in Europe or are used as vaccine viruses there. The isolates could be differentiated into seven groups based on restriction patterns. The largest group contains isolates from passeriform and psittacine birds and could further be subdivided into four subgroups. Two other groups are represented by herpesvirus isolates of quail and crane, and by isolates of pigeon and owl. Duck plague virus, herpesvirus of turkey, infectious laryngotracheitis virus and a herpesvirus isolate from tragopan all exhibited distinct restriction patterns. In general, our results parallel earlier cross-neutralization studies and yield additional, more detailed information on the relationship between different herpesvirus isolates of birds.

Fatal avian polyomavirus infection during quarantine in adult wild-caught red-faced lovebirds (Agapornis pullaria).

An outbreak of avian polyomavirus infection is reported in a group of six wild-caught red-faced lovebirds (Agapornis pullaria), all of which died during quarantine. The birds had not shown any previous symptoms. Histologic examination of the lungs, kidneys, livers, and spleens revealed the presence of basophilic intranuclear inclusions. Avian polyomavirus was isolated from the liver and the spleen. Neutralizing antibodies were detected in the sera from other lovebirds (Agapornis personata, Agapornis taranta) that had contact with the A. pullaria. A serologic comparison showed a close relationship with budgerigar polyomavirus.

Investigation on the frequency of Chlamydia sp. infections in tits (Paridae).

A total of 788 cloacal and pharyngeal swabs were taken from 399 free-living, clinically healthy tits. Ten nestlings were examined by cloacal swabs only. Additionally, six dead tits were necropsied and various organs were collected for testing. All swabs and organ samples were tested for Chlamydia Chlamydia sp. was detected by immunofluorescence with FITC-conjugated monoclonal antibodies after 90 h incubation on Buffalo-Green-Monkey (BGM) cell cultures at 37 degrees C. Fifty-four per cent of 399 tits examined were Chlamydia positive. Chlamydia was detected in 154 of 399 pharyngeal swabs (39%) and in 144 of 389 cloacal swabs (37%). Blue tits (Parus caeruleus) were most frequently Chlamydia positive (70%), followed by great tits (Pants major) with 53% and marsh tits (Parus palustris) with 38%. No significant relationship was established between Chlamydia detection and time of the year, age, sex or size of birds at the time of sampling. Tits examined two or three times were intermittent shedders. Four of the 10 tit nestlings (Parus major) examined were already Chlamydia positive at the age of 10 days. Chlamydia was detected in four of six necropsied tits. The necropsies showed that all of the positive tits were cachectic. The livers of three of the four positive tits were pathologically changed and splenomegaly was observed in two of the four. No CEF (chick embryo fibroblast)-pathogenic viruses and no bacteria were found in these animals. This indicates that chlamydiosis was the only cause of death. Two of the Chlamydia sp. isolates from the dead tits produced cytopathic effects (cpe) in BGM cell cultures. These cytopathic isolates may be more virulent than the isolates from healthy tits, which did not cause visible alterations in BGM cell cultures. The results of this study and the frequent shedding of Chlamydia in healthy tits prove that most tits intermittently shed Chlamydia sp. as latent carriers, but that lethal Chlamydia infections can also occur in tits.

Antigenic diversity of psittacine herpesviruses: cluster analysis of antigenic differences obtained from cross-neutralization tests.

A total number of 28 viruses were isolated from different diseased or dead psittacine birds on various occasions. All of them were end-point purified and identified as herpesviruses (HV) on the basis of chloroform sensitivity, inhibition of multiplication in cell cultures by 5 iodo 2'-deoxyuridine (IUDR), lack of haemagglutination and morphology by electron microscopy. Antisera were produced in rabbits with each of the viruses. Herpesviruses from other birds (duck plague HV, stork HV, crane HV) were used for comparison. All virus/antiserum combinations were tested for homologous and heterologous neutralization in chick embryo fibroblast (CEF) cultures. The r1 and r2, as well as R-values were calculated according to the formula of Archetti & Horsfall (1950). These values formed the basis of the production of minimal trees and dendrograms. None of the non-psittacine viruses were neutralized with antisera produced against any of the psittacine herpesviruses and vice versa. Within the psittacine viruses three distinguishable subtypes were established. It is proposed to use as representative HV for subtype 1, 2 and 3 the strains KS 144/79, 6840/87 and 3115/88, respectively. Isolates 1483/92 and 8326/87 were distinguishable from each other and all other isolates; they are candidates for possible subtypes 4 and 5.