Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus.

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting.

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.

Sequence variation and conservation in virulence-related genes of Bordetella pertussis isolates from the UK.

To determine the value of gene markers for surveillance and to assess the genetic stability of potential acellular pertussis vaccine components, the sequence variation in ten virulence-related genes of Bordetella pertussis was investigated in strains isolated in the UK between 1920 and 2002. These genes encode: pertactin (prnA); pertussis toxin subunits S1 (ptxA) and S3 (ptxC); tracheal colonization factor (tcfA); bordetella autotransporter protein C (bapC); bordetella resistance to killing protein (brkA); fimbrial antigen 2 (fim2); outer-membrane protein Q (ompQ); virulence-activated gene 8 (vag8) and adenylate cyclase toxin (cyaA). The encoded proteins are either components of current acellular vaccines (ACVs), or potential virulence markers for B. pertussis. Three strains used in the pertussis UK whole-cell vaccine (WCV), strain Tohama-I used for production of ACV components and the type strain of B. pertussis (18323(T)) were also analysed. Several novel alleles were found. The UK isolates were assigned multi-locus sequence types (MLSTs) according to a previously described scheme for B. pertussis based on three of these genes (ptxA, ptxC and tcfA). Compared with isolates from other countries, the UK clinical strains showed a distinct distribution of MLSTs. Apart from one strain that was MLST-3, all other recent isolates (2000-2002) were identified as MLST-5. These isolates differed from the three WCV strains, which were MLST-2 or MLST-3, the Tohama-I strain (MLST-2) and the type strain of B. pertussis (MLST-9). MLST-3 and MLST-5 differ only by a single synonymous mutation, but this method does indicate that currently circulating strains of B. pertussis are not identical to the vaccine types, and they may differ in other important characteristics. Two new MLSTs were identified amongst historical UK isolates. Sequence-based typing offers a convenient method of analysing and comparing populations of B. pertussis from different time periods and from different countries. The variation exhibited by prnA and fim2 suggests that they could be useful, additional epidemiological markers in such a typing scheme.