The 2000 canine distemper epidemic in Caspian seals (Phoca caspica): pathology and analysis of contributory factors.

More than 10,000 Caspian seals (Phoca caspica) were reported dead in the Caspian Sea during spring and summer 2000. We performed necropsies and extensive laboratory analyses on 18 seals, as well as examination of the pattern of strandings and variation in weather in recent years, to identify the cause of mortality and potential contributory factors. The monthly stranding rate in 2000 was up to 2.8 times the historic mean. It was preceded by an unusually mild winter, as observed before in mass mortality events of pinnipeds. The primary diagnosis in 11 of 13 seals was canine distemper, characterized by broncho-interstitial pneumonia, lymphocytic necrosis and depletion in lymphoid organs, and the presence of typical intracytoplasmic inclusion bodies in multiple epithelia. Canine distemper virus infection was confirmed by phylogenetic analysis of reverse transcriptase-polymerase chain reaction products. Organochlorine and zinc concentrations in tissues of seals with canine distemper were comparable to those of Caspian seals in previous years. Concurrent bacterial infections that may have contributed to the mortality of the seals included Bordetella bronchiseptica (4/8 seals), Streptococcus phocae (3/8), Salmonella dublin (1/8), and S. choleraesuis (1/8). A newly identified bacterium, Corynebacterium caspium, was associated with balanoposthitis in one seal. Several infectious and parasitic organisms, including poxvirus, Atopobacter phocae, Eimeria- and Sarcocystis-like organisms, and Halarachne sp. were identified in Caspian seals for the first time.

Development of reverse transcription-PCR (oligonucleotide probing) enzyme-linked immunosorbent assays for diagnosis and preliminary typing of foot-and-mouth disease: a new system using simple and aqueous-phase hybridization.

A reverse transcription-PCR (RT-PCR)-enzyme-linked immunosorbent assay system that detects a relatively conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease virus (FMDV) has been developed. The high specificity of the assay is achieved by including a rapid hybridization step with a biotin-labeled internal oligonucleotide. The assay is highly sensitive, fast, and easy to perform. A similar assay, based on a highly variable region of the FMDV genome and employing a single asymmetric RT-PCR and multiple hybridization oligonucleotides, was developed to demonstrate the method's ability to type FMDV. Based on our theoretical and practical knowledge of the methodology, we predict that similar assays are applicable to diagnosis and strain differentiation in any system amenable to PCR amplification.

Localization of foot-and-mouth disease virus RNA by in situ hybridization within bovine tissues.

Foot-and-mouth disease is a highly contagious disease of cloven hooved animals. In cattle, both acute and long-term persistent infections occur. Foot-and-mouth disease virus (FMDV), a picornavirus, has been shown, using virus isolation procedures, to replicate in the pharynx and soft palate of cattle. In this study, in situ hybridization has been used to detect FMDV RNA within the cells of tissues removed from infected bovines. A digoxigenin-labelled anti-sense RNA probe was prepared corresponding to a region of the FMDV genome encoding part of the RNA-dependent RNA polymerase (3D). The efficacy and specificity of this probe for in situ hybridisation was determined using virus-infected cells in tissue culture. Strong cytoplasmic staining was only detected in FMDV-infected cells. Various tissue samples were collected from FMDV-infected cattle between 5 and 17 days post-infection. Viral RNA was detected by in situ hybridisation within cells of the soft palate, tonsil and pharynx up to 17 days post-infection. This technique is useful for the study of FMDV localization in cattle both during and after the acute clinical phase of disease and may assist in identifying specific sites of virus persistence.

Antibody to the nonstructural proteins of foot-and-mouth disease virus in vaccinated animals exposed to infection.

Cattle which have been infected with foot-and-mouth disease (FMD) virus can be differentiated from those that have been vaccinated on the basis of the detection of antibody to one or more of the non-structural (NS) proteins of the virus. Cattle which have been protected by vaccination can become persistently infected with FMD virus (FMDV) without ever showing clinical signs. Vaccinated, protected cattle which are persistently infected cannot be distinguished from animals that merely have been vaccinated on the basis of serological tests for antibody to the structural proteins of FMDV. Sera were collected from groups of cattle for varying periods after exposure to infection under experimental conditions. On the basis of isolation of virus from probang samples collected during the course of the experiments it was possible to classify the cattle according to the following criteria; naive, infected and eliminated the virus (convalescent), infected and persistently infected with FMDV (carriers), vaccinated alone, vaccinated and either convalescent or carrier. Sera were examined for antibody to the NS proteins Lb, 2C, 3A, 3D, and 3ABC by an indirect profiling ELISA using E. coli-expressed fusion proteins as antigens. Considerable variation was observed in the antibody response to NS proteins of both naive and vaccinated animals following infection. The extent of individual variation was so great that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined. The majority of vaccinated, protected animals showed an antibody response to NS proteins, particularly 3ABC, following exposure to infection. However, the carrier state was demonstrated in some vaccinated, protected animals in which no antibody response to any of the NS proteins could be detected. The detection of antibody to NS proteins can therefore be used on a group, or herd, basis to detect circulation of virus in a vaccinated population but further investigations in the field are required to determine the sampling level necessary for statistical acceptance. On an individual animal basis, however, freedom from antibody to NS proteins in a vaccinated animal, or an animal of unknown history, does not necessarily imply that the animal is free from infection with FMD virus and, furthermore, the titre of antibody to NS proteins is not a useful predictive measure of whether or not an infected animal has successfully eliminated the virus.

Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease.

A reverse transcription polymerase chain reaction (RT-PCR) method was compared with virus isolation in cell culture and the antigen detection ELISA for the primary diagnosis of foot-and-mouth disease (FMD) on 166 clinical samples from the field. Eighty samples were positive by virus isolation/ELISA and 78 by RT-PCR. The RT-PCR detected FMD viral RNA in 11 of the 86 samples assessed as negative by virus isolation/ELISA but conversely failed to diagnose 13 samples identified as positive by the latter procedures. This RT-PCR is not serotype-specific so a cDNA product is indicative of the presence of FMD viral RNA only. Confirmation of the specificity of the cDNA product and the identification of the serotype requires nucleotide sequence analysis. The value of the RT-PCR is that it can rapidly facilitate the molecular analysis of field isolates and thus provide important epidemiological information regarding the source of outbreaks. However, it is a sophisticated technique requiring specialised equipment, expertise and refined reagents and has to be used in conjunction with current procedures for FMD diagnosis.

Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA.

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.

Geographic distribution and epidemiology of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is an important viral disease of goats and sheep prevalent in West Africa and the Middle East. In recent years, PPR has emerged in India, first in the South India and later in North India. To study the genetic relationships between viruses of distinct geographical origin we have sequenced a 322 nucleotide cDNA fragment of the fusion protein gene generated using reverse transcription followed by polymerase chain reaction (PCR) amplification. Viruses from nineteen independent PPR outbreaks were compared; these included the prototype African strain from Senegal and viruses from disease outbreaks which have occurred at different times and locations across Africa, Arabia, the Near East and the Indian subcontinent. Four separate lineages of the virus were identified and the virus isolates from Asia over the past 2 years were all of one lineage which had not previously been identified in Africa or Asia.

Evaluation of polymerase chain reaction for the detection and characterisation of rinderpest and peste des petits ruminants viruses for epidemiological studies.

The high sequence variability found in RNA viruses makes it difficult to design primers for reverse transcription-polymerase chain reaction amplification which will be certain to work with all new field isolates. To overcome this problem for the detection and differential diagnosis of rinderpest (RP) and peste des petits ruminants (PPR) viruses (V), we have designed several sets of primers, based on well-conserved sequences in the P and F genes. Analysis of a large number of field isolates from every region of the world where RPV and PPRV are found showed that no sample failed to react with more than one of the primer sets. To facilitate the multiple analyses, the reverse transcription step was performed using random hexanucleotide primers and aliquots of the cDNA were then amplified using a panel of primer sets to identify and differentiate between the virus nucleic acids in the samples. Evaluation of the method was carried out using eye swabs collected from cattle experimentally infected with RPV and goats infected with PPRV during the course of vaccine trials and on field samples such as whole blood, mouth swabs, lung, spleen and other tissues submitted to the laboratory for diagnosis. Sequencing the PCR products enabled us to examine the genetic relationships between new and previous field isolates from different geographical areas.