Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: implications for Lyme disease vaccine and diagnostic assay design.

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Potential of a recombinant Schistosoma bovis-derived glutathione S-transferase to protect cattle against experimental and natural S. mattheei infection.

The potential of a recombinant Schistosoma bovis-derived glutathione S-transferase (rSb28GST) to protect cattle against S. mattheei infection was tested in Zambia. All animals were challenged 2 weeks after the second inoculation with either 0.250 mg rSb28GST in adjuvants (vaccinated calves, n = 14) or adjuvants alone (controls, n = 14). In a first experiment, 7 vaccinated and 7 control animals were exposed to 10000 S. mattheei cercariae percutaneously. All animals developed clinical schistosomiasis 7-8 weeks after challenge. At perfusion, 12 weeks after challenge, vaccinated and control groups had averages of 887 and 541 eggs per gramme (epg) faeces, 6515 and 5990 worms, and 4.2 and 3.4 million tissue eggs, respectively. These results indicate that the immunization protocol used did not protect these calves against the massive single experimental challenge. In a second experiment, another 2 groups (n = 7) of vaccinated and control animals were challenged naturally over a period of 9 months on a farm known to be endemic for S. mattheei. The natural infections were much lighter in intensity, as indicated by the mean faecal egg count (13 epg), worm count (139) and tissue egg count (294000) in non-vaccinated controls. In vaccinated calves, significant reductions in female worm burdens (50%), faecal egg count (89%) and miracidial counts (93%) were recorded. Total tissue egg counts were also reduced by 42% in vaccinated animals. It therefore appears that the rSb28GST can provide significant protection in cattle against S. mattheei under conditions of low to moderate natural infection.

Comparison of the persistent activity of ivermectin, abamectin, doramectin and moxidectin in cattle in Zambia.

The persistent efficacy of four commercially available macrocyclic lactones (ML) in maintaining reduced faecal egg counts in cattle grazing naturally infested pastures was evaluated in 44 zebu animals aged 1-2 years in Zambia. The study started in February (rainy season) when the strongyle egg output was increasing. Four days before the start of the trial, all animals were treated with a double dose of oxfendazole. They were then divided into five groups which were again treated on day 0. Groups A, D, I and M received 0.2 mg kg-1 of abamectin, doramectin, ivermectin and moxidectin, respectively. Animals of group C received albendazole (7.5 mg kg-1). Faecal samples were collected twice a week for egg counts and larval differentiation. Faecal egg counts in the C group increased from day 21 onwards and plateaued from day 42 between 180 and 380 eggs per gram. The main genera found in cultures were Cooperia (90%) and Haemonchus (7%). Faecal egg excretion in groups M, A, D and I started on day 35, 42, 42 and 45, respectively. Subsequently and until day 84, average counts in these four groups were always significantly lower than in group C. Compared with albendazole, all four ML gave over 95% reduction in cumulative faecal egg counts for 42 days after treatment. The percentage efficacy was still over 84% by day 84 when an average cumulative egg count of 11320 eggs per gram faeces was calculated in group C. In addition, there was no significant difference in efficacy between the four ML groups at any of the sampling dates. During the trial no significant difference in weight gain between any of the groups was observed.