Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.     

Genotypic characterisation of Giardia from domestic dogs in the USA

The first large-scale urban survey of Giardia infections in dogs was undertaken in the USA. It involved several locations in the Western United States with Giardia isolates from microscopy-positive samples characterised by multi-locus PCR and sequencing. A high prevalence of Giardia was confirmed in asymptomatic domestic dogs, and for the first time, provides evidence that zoonotic assemblages/subgroups of Giardia occur frequently in domestic dogs living in urban environments, and more frequently than the dog specific assemblages.     

Genotypic characterisation and cluster analysis of Campylobacter jejuni isolates from domestic pets, human clinical cases and retail food

The genetic similarity of Campylobacter jejuni isolates from pets, compared to human clinical cases and retail food isolates collected in Ireland over 2001-2006 was investigated by cluster analysis of pulsed-field gel electrophoresis (PFGE) fingerprinting profiles. Comparison of the PFGE profiles of 60 pet isolates and 109 human isolates revealed that seven (4.1%) profiles were grouped in clusters including at least one human and one pet C. jejuni isolate. In total six (1.6%) of 60 pet and 310 food profiles were in clusters with at least one food and one pet C. jejuni isolate. The detection of only a small number of genetically indistinguishable isolates by PFGE profile cluster analysis from pets and from humans with enteritis in this study suggests that pets are unlikely to be an important reservoir for human campylobacteriosis in Ireland. However, genetically indistinguishable isolates were detected and C. jejuni from pets may circulate and may contribute to clinical infections in humans. In addition, contaminated food fed to pets may be a potential source of Campylobacter infection in pets, which may subsequently pose a risk to humans.     

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.     

Induction of diplodiosis, a neuromycotoxicosis, in domestic ruminants with cultures of indigenous and exotic isolates of Diplodia maydis

Diplodiosis, a neuromycotoxicosis, principally of cattle, which is characterized by ataxia, paresis and paralysis, was induced in 13 cattle, 16 sheep and 3 goats, by dosing them with Diplodia maydis [= D. zeae (Schw.) Lév.] cultured on sterilized maize seeds. The results of these experiments confirmed the findings of earlier workers that diplodiosis is a mycotoxicosis caused by D. maydis. The intoxication was induced with cultures of South African isolates of D. maydis obtained from local maize, one of which was associated with a suspected field outbreak, and with cultures of isolates from maize imported from the United States of America and Argentina. Other findings emerging from the experiments were, inter alia, that cultures incubated for less than 8 weeks were seemingly non-toxic, that there was little individual variation in response of cattle to cultures of the different toxic isolates or batches of the isolates, that apparent relapses of clinical signs can occur several weeks after dosing had ceased and that a small percentage of animals can show permanent locomotory disturbance. Light microscopical examination revealed no lesions in acutely affected animals, but an extensive laminar subcortical status spongiosis was evident in the cerebrum and cerebellum of a sheep that had been long paralysed and a steer that had permanent locomotory disturbance.     

Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in Brazil

The purpose of the present study was the genetic characterization, sequencing and phylogenetic analysis of 18S rDNA sequences of Cryptosporidium isolates obtained from different animal hosts in Brazil. Fecal samples containing Cryptosporidium oocysts were obtained from chickens, ducks, quails, guinea pigs, dairy calves, dogs and cats. For amplification of 18S rDNA sequences the Secondary-PCR product of the extracted DNA from fecal suspension of each studied animal was utilized. The primary genetic characterization of Cryptosporidium sp. was performed using RFLP with the enzymes SspI and VspI. DNA samples were sequenced and subjected to phylogenetic analysis. The results showed C. baileyi infecting two ducks and one quail and C. melagridis infecting one chicken. The sequences obtained from Cryptosporidium sp. infecting guinea pigs were not identified within groups of known Cryptosporidium species. The isolates found parasitizing cats and one dog were diagnosed as C. felis and C. canis, respectively. One isolate of calf origin was identified as C. parvum. The phylogenetic analysis showed clear distribution of isolates between two Cryptosporidium sp. groups according to their gastric or intestinal parasitism. A great genetic distance was observed between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. The results obtained during this study constitute the first report of rDNA sequences from C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum isolated in Brazil.     

Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination

Objective To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.
Design A comparative study of polymerase chain reaction genotypes in different populations of B bovis .
Procedure Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.
Results No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.
Conclusion No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.     

Diversity and stability of Aleutian mink disease virus during bottleneck transitions resulting from eradication in domestic mink in Denmark

Aleutian mink disease (plasmacytosis) virus (AMDV) in domestic mink (Neovison vison) has been subject to eradication in Denmark since 1976. In 2001, approximately 5% of Danish mink farms were still infected and all were located in the northern part of the peninsula of Jutland. In the present study a total of 274 Danish isolates of AMDV collected during the two seasons of 2004 and 2005 were characterized by partial sequencing of the coding region of the non-structural (NS) proteins. Older AMDV isolates from Denmark, available, were also included. The Danish isolates represent a very homogenous cluster compared with Swedish, Finnish and Dutch isolates and seem to represent a minor fraction of the genetic diversity previously found in Denmark. Stability of nucleotide deviations reveals that the purifying selection of bottlenecks imposed on the AMDV population in Denmark by the stamping out policy for more than 6 years exceeds the rate of mutation driven diversity. Among the isolates from farms in northern Jutland two distinct types could be identified and within each of them a number of sub-types which were all useful in tracking spread of infections. Infection at a farm the preceding season was a predisposing risk parameter for disease outbreak at a farm, and strain identity substantiates the suggestion that inadequate disinfection is involved in the recurrence of outbreaks. In cases of new introductions to farms it is indicated that contact including transport between farms played a most significant role.     

A survey of antimicrobial resistance in Salmonellae isolated from animals in England and Wales during 1984-1987

Resistance to 14 antimicrobial substances was tested in 18,647 salmonella strains isolated from animals, their environment and from animal feeds during the period 1984-1987. Of the 2284 Salmonella dublin strains the percentage sensitive to all the antimicrobial substances ranged from 18.1 to 26.8. Resistance to the higher concentration of streptomycin (S25) ranged from 1.9 to 6.4%, whereas the corresponding figures when the lower concentration (S10) was used were 32.3 and 63.8%. Resistance to the higher sulphonamide concentration (Su500) never exceeded 3.3%, although in 1987 70.3% of strains showed resistance to the lower concentration. In general, less than 1% of strains showed resistance to the other antibacterial substances. No strains resistant to amikacin (AK), apramycin (Apr), gentamicin (CN) and colistin (CT) were detected. Of the 8677 S. typhimurium strains the percentage sensitive to all the antimicrobial agents ranged from 6.6 in 1985 to 13.6 in 1987. Resistance to tetracycline (T), ampicillin (PN), trimethoprim (TM) and chloramphenicol (C) ranged from 36.5 to 58.8%, the highest percentages being detected with tetracyclines. Less than 1% of strains showed resistance to furazolidone and none was resistant to amikacin and colistin. Resistance to apramycin ranged from 3.1% in 1984 to 11.6% in 1985; the figures for gentamicin were approximately half that of apramycin. In 1984, 41.6% of strains showed resistance to neomycin but only 8.5% in 1987. The fall in neomycin resistance was associated with the epidemic spread of the commonest phage-type DT204C becoming sensitive to neomycin. Of the 7687 strains of serotypes other than S. typhimurium and S. dublin the percentage sensitive to all antimicrobial agents ranged from 23.7 in 1985 to 14.7 in 1987. Resistance to tetracyclines and sulphonamides (Su500) ranged from 5.2 to 12.1% and 5.8 to 13.3% respectively. Resistance to the other antimicrobial agents was usually less than 5%.     

Antigenic variation among foot-and-mouth disease virus type A field isolates of 1997-1999 from Iran

The sequences of the antigenically relevant capsid proteins VP1-3 of 10 isolates obtained during an epizootic of serotype A foot-and-mouth disease virus in Iran, and collected within two and a half years, were found to be highly similar. However, each isolate differed by at least one amino acid from all others. This prompted us to analyze the immunological reactivity of the isolates. To this end, monoclonal antibodies (mAbs) against one isolate were generated and characterized with regard to neutralizing activity and reactivity with trypsinized virus. These mAbs as well as others raised against A22 virus were used for antigen profiling. This distinguished four antigenic conditions among the isolates and 16 reactivities among the mAbs. These findings, together with the observed sequence differences indicated the location of several epitopes. Many mAbs recognized the minor antigenic sites on VP2 and 3 and some the major site, the GH-loop of VP1. One epitope was composed of residues of the capsid proteins VP1 and 2.     

Assessment of three methods for multilocus fragment typing of Cryptosporidium parvum from domestic ruminants in north west Spain

The performance of three different methods, capillary electrophoresis (CE), high resolution slab-gel electrophoresis and sequencing, for PCR fragment size analysis of two Cryptosporidium parvum microsatellite regions, ML1 and ML2, was investigated by analysing 27 isolates from calves and 14 from lambs. To assess genetic variability of this protozoan in domestic ruminants in north west Spain, results were combined with sequence analysis of the 60 kDa glycoprotein (GP60) gene creating a multilocus type and analysed by farm and host species. CE showed greater overall typability (T), discriminatory power and ease of use than slab-gel electrophoresis and sequencing which were both affected by PCR stutter, especially at ML2. CE fragment sizes were consistently 4 bp longer compared to sequencing which is considered the gold standard for allele sizing but which gave the lowest typability; CE sizes were therefore adjusted. Only three alleles were identified at the ML1 locus (ML1-238, ML1-229 and ML1-226). The ML2 locus was more polymorphic and eight alleles were found (ML2-235, ML2-233, ML2-231, ML2-229, ML2-227, ML2-225, ML2-201 and ML2-176). Adjusted ML1 and ML2 CE fragment sizes were combined with GP60 subtype for 37 of the 41 C. parvum isolates which were typable at all three loci (T=0.90): nine multilocus types (MLTs) were identified. The discriminatory power of the 3-locus typing method was 0.83. Greater genetic variability was observed in calf isolates (7 MLTs) than in those from lambs (4 MLTs) although more calf isolates were studied. The most common MLT in cattle was MLT1 (ML1-238, ML2-231, GP60 subtype IIaA15G2R1), while MLT3 (ML1-238, ML2-227, GP60 IIaA16G3R1) was predominant in lambs. Our findings demonstrate that high discrimination can be achieved by means of multilocus typing. CE appears to be an economic and rapid option for performing microsatellite fragment size analysis offering good typability, discrimination and ease of use but may require calibration to sequenced standards.