Masanobu Kamada

Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge

Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration. Ten of the 12 foals were inoculated with 10(4)-10(10) colony forming units (cfu) of ATCC 33701 strain. The other 2 foals were inoculated with 10(9) cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P-). All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5-13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia. The two foals challenged with the ATCC 33701P- strain showed neither clinical signs of disease nor gross lesions. Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Lack of virulence of the murine fibroblast adapted strain, Kentucky A (KyA), of equine herpesvirus type 1 (EHV-1) in young horses

The virulence of the cell culture adapted KyA strain of equine herpesvirus type 1 (EHV-1), which lacks at least six genes by deletions in its genome, was assessed by intranasal inoculation of six young horses that were serologically negative for EHV-1. No horses showed clinical signs, and a neutralizing antibody response against EHV-1 was detected in two horses which had antibodies against EHV-4 prior to the inoculation. A challenge experiment using a highly virulent strain of EHV-1 conducted 4 weeks later against 4 of the 6 horses inoculated intranasally with the KyA strain and 2 control horses revealed that (i) the KyA inoculated horses were protected from manifestation of clinical signs detected in both control horses, with the exception of pyrexia, (ii) duration of virus isolation from the KyA inoculated horses after the challenge was remarkably shortened as compared to that from control horses; (iii) thus, animals inoculated with the KyA and challenged with pathogenic EHV-1 showed a reduction in the time of virus shedding and viremia; (iv) two horses which exhibited no antibody responses after the KyA inoculation showed antibody responses after the challenge significantly higher than those of control horses. The results reveal that the KyA strain has no virulence but still possesses immunogenicity for horses, suggesting that some of the genes deleted from the KyA strain might have importance in the expression of EHV-1 virulence.

Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals

The diagnostic value of tracheal aspiration was evaluated through comparison with other diagnostic methods using an experimental model of Rhodococcus equi (R. equi) pneumonia in foals. Pneumonia was induced by spraying of the virulent R. equi strain ATCC 33701 into the trachea of foals. All foals developed fever from 11 to 16 days after bacterial inoculation. One foal was euthanized on day 26 due to its poor prognosis, and other foals euthanized on day 43. During the experiment, some tests for diagnosis of Rhodococcus equi pneumonia such as tracheal aspiration, radiography, serodiagnosis and fecal culture were carried out. R. equi was continually isolated from tracheal aspirates collected via a silicone catheter inserted transnasally on day 8 to day 32 after bacterial inoculation. On the other hand, radiography, serodiagnosis and fecal culture were demonstrated to be valuable diagnostic methods, but to be limited compared with tracheal aspiration. Indirect fluorescent antibody technique (IFA) using a monoclonal antibody against the 15- to 17-kDa virulence-associated antigens (VapA) of R. equi and PCR targeting the structural gene of VapA detected bacteria in tracheal aspirates less sensitively than the isolation technique although they were more rapid. Therefore, we conclude that a combination of tracheal aspiration and bacterial isolation was the most valuable method for routine diagnosis of R. equi pneumonia in foals.

Pathology of equine pneumonia associated with transport and isolation of Streptococcus equi subsp. zooepidemicus.

Seven horses that died of pneumonia associated with transport yielded Streptococcus equi subsp. zooepidemicus (S.z.) from their pulmonary lesions. These lesions were divisible roughly into two types, serous haemorrhagic pneumonia and multiple foci of coagulative necrosis, which were considered to reflect a temporal difference in the process of lesion formation. Immunohistologically, S.z. antigen was detected in both types of lesion. Acute necrotic lacunar tonsillitis was considered to play an important role in the onset of the pneumonia.

Electropherotypes, serotypes, and subgroups of equine rotaviruses isolated in Japan.

SummaryElectropherotypes (ET), serotypes, and subgroups of equine rotaviruses isolated from foals in Japan were determined. The ETs of 136 isolates from 1981 through to 1991 were divided into six groups: ET-A-ET-F. The ET-A,-B, -C, -D, -E, and -F were present in 3, 1, 121, 9, 1, and 1 strains, respectively. Representative viruses of ET-A, -B, -C, and -D were identified as serotype G 3. Viruses of ET-E and -F were identified as serotypes G 10 and G 5, respectively. The four representative viruses of serotype G 3 did not belong to either subgroup I or II. The two viruses of serotypes G 5 and G 10 belonged to subgroup I. Serotype G 3 strains possessing ET-C were prevalent among the foals throughout the study period.

Phylogenetic analyses of the matrix and non-structural genes of equine influenza viruses

SummaryMatrix (M) and nonstructural (NS) genes of thirteen equine H3N8 and H7N7 influenza viruses were sequenced and analyzed from an evolutionary point of view. The M and NS genes of H3N8 viruses isolated between 1989 and 1993 evolved into two minor branch clusters, including isolates from Europe and the American continent, respectively. It was noteworthy to reveal that the nucleotide sequences of the M and NS genes of an earlier American strain showed highest homology to those of recent European viruses. “Frozen evolution” was observed in the M and NS genes of A/eq/LaPlata/1/88. It was also evident that the NS gene of an H7N7 virus from 1977 was very similar to that of a 1979-H3N 8 virus, while the M gene was closest phylogenetically to that of the earliest H7N7 virus isolated in 1956. Furthermore, the M2 protein of A/eq/Newmarket/1/77 virus contained a carboxyl terminal deletion of three amino acids. The evolutionary rates of the M and NS genes of H3N8 equine influenza viruses were estimated to be 5.4 × 10−4 and 5.1 × 10−4 substitutions per site per year, respectively, which were slower than those of human viruses.

Comparative sequence analysis of the inverted terminal repetition in the genomes of animal and avian adenoviruses.

The nucleotide sequences at the inverted terminal repetitions from two animal adenoviruses, infectious canine hepatitis virus and equine adenovirus, and from one avian adenovirus, CELO, were analyzed. DNAs from infectious canine hepatitis virus and equine adenovirus contain a homologous region which is 23 nucleotides long from the terminus. The first 17 nucleotides of this region are identical to the ones in human adenovirus type 2 DNA. The striking homologous sequence of 14 nucleotides, conserved in the inverted terminal repetitions of several human adenovirus strains and in simian adenovirus type 7, is only partially conserved in the two animal and one avian adenoviruses reported here.

The complete sequence of African horsesickness virus serotype 4 (vaccine strain) RNA segment 5 and its predicted polypeptide compared with NS1 of bluetongue virus.

The complete sequence of RNA segment 5 of the African horsesickness virus serotype 4 (AHSV-4) vaccine strain was determined from cDNA clones inserted into pBR322. The RNA is 1751 bp long (M(r) 1.12 x 10(6)) and contains an open reading frame encoding a protein of 548 amino acids (M(r) 63,122) with a net charge of +0.5 at neutral pH. A comparison of the sequence of AHSV-4 segment 5 with that of segment 6 of bluetongue virus (BTV) serotypes 10 and 17 revealed 49.2% and 48.9% nucleotide similarity, respectively, and 31.4% amino acid similarity. However, AHSV-4 segment 5 has no significant similarity to BTV segment 5. In addition, Northern blot hybridization showed that full-length AHSV-4 segment 5 cDNA cross-hybridized with the corresponding genes of all serotypes of attenuated AHSV.

Detection of virulent Rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of R. equi pneumonia in foals

Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest level. Virulent R. equi were isolated from the feces of the foals at a high frequency and from their environmental soil on the farms. Evidence that serum antibody response to 15- to 17-kDa antigens of virulent R. equi occurred naturally in every foal in correlation with the quantitative changes of fecal R. equi during the first 1 to 3 months of life suggests that intestinal virulent R. equi might be the most important source of antigenic stimulation in foals from contaminated farms.