A molecular epidemiological study on Haemorrhagic septicaemia disease in Pakistan

Ahmed Moustafa

Haemorrhagic septicaemia (HS) is an acute fatal septicaemic disease of cattle and buffaloes associated with strains of serotypes B:2 and E:2 of the bacterium Pasteurella multocida. Asia and Africa are currently the regions where HS occurs with the highest prevalence and has the greatest economic importance. There is currently only limited information available on the diversity of P. multocida strains that cause HS in these regions. A retrospective epidemiological case-control study was performed in Karachi, Pakistan from January to April 2013 looking at HS cases that occurred in the 2012 calendar year. The owners from 217 dairy cattle and dairy buffalo farms from six different locations around Karachi were interviewed. The study was based on a questionnaire that was designed to identify independent variables that were statistically associated with the presence of HS on the farm. The final multivariable logistic model contained five factors. Two protective factors were identified: HS vaccination (Odds Ratio (OR) = 0.22) and the length of time cattle were kept on farm (months): for every extra month cattle were kept, the odds of disease were reduced by a factor of 0.9. In contrast, supplying underground water and the presence of foot and mouth disease on the farm increased the risk of infection by 2.90 and 2.37, respectively. In addition, for every extra animal on the farm, the risk of infection increased by a factor of 1.01. To understand the epidemiology of HS in Karachi dairy herds more fully, further in-depth research is required to study the risk and protective factors identified in this survey and to evaluate risk mitigation strategies where possible. The study also showed the need for developing a point-of-care diagnostic test that can be used by veterinarians to diagnose the disease on the farm. Haemorrhagic septicaemia-associated strains of P. multocida were then analysed using molecular methods. Haemorrhagic septicaemia vaccine strains and HS-associated field isolates were obtained from different places in Pakistan and as a comparison group, vaccine strains and HS-associated field strains were obtained from Thailand to investigate the genetic diversity of strains associated with the disease from two endemic countries. Initially, 21 field isolates and three vaccine strains from different regions within these countries were analysed by multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). The MLST technique was not able to differentiate between the strains from Pakistan and Thailand as all of the tested isolates (n = 21) were sequence type (ST) 122. The PFGE technique showed a difference of one band between the Thai and Pakistani isolates. Neither technique was able to show any variation between isolates from the same country. Based on the MLST and PFGE results, 12 of the 24 HS-associated strains were selected for next generation sequencing (NGS). Analyses of the genome data identified a core set of 1824 genes that were shared by the 12 HS-associated Asian strains, M1404 (North American HS-associated strain), and the available P. multocida complete genomes of strains Pm70, 3480, 36950 and HN06. Four sets of unique genes were found. One set (96 genes) was shared by all HS-associated strains. The second set (59 genes) was shared by the Asian HS-associated strains only. The third set (39 genes) was shared by seven out of nine Pakistani HS-associated strains. The last set (42 genes) was shared by the remaining two Pakistani HS-associated strains that were studied. The set of 96 unique genes, found in all HS-associated strains but absent from the non-HS-associated strains Pm70, 3480, 36950 and HN06, was identified by the PHAST bacteriophage detection algorithm to encode two putative temperate phages. It seems reasonable to suspect that the presence of these putative prophages may provide virulence genes that contribute to the pathogenesis of HS. The set of 59 unique genes shared by the 12 Asian HS-associated strains but not the North American HS-associated strain M1404, was also predicted to encode a temperate phage. Likewise, another putative temperate phage was predicted in the seven Pakistani strains that accounted for the set of 39 unique genes they shared. Interestingly, two Pakistani strains (BUKK and TX1) carried acquired antimicrobial resistance genes as predicted by the Resfinder tool. BUKK and TX1 strains shared 42 unique genes that were not present in any other HS strains. Some of these unique genes may contribute to a putative integrative conjugative element (ICE). The putative ICE of BUKK and TX1 is not identical to ICEPmu1, the first ICE that was found in P. multocida (strain 36950), and therefore, it may be the second ICE to be discovered in P. multocida strains. Phylogenetic analysis, predicted by analysis of core genome single nucleotide polymorphisms, demonstrated a strong correlation between the individual strains and their countries of origin. The Pakistani and Thai strains were more closely related to each other than the North American strain. Similarly, the isolates from Pakistan clustered together, and were distinctly separate from Thai isolates. A specific rapid diagnostic test, based on loop-mediated isothermal amplification (LAMP), for HS-associated B:2 strains, was developed using the previously-identified 96 unique genes obtained from NGS. Duplicates of each reaction were run, and this allowed two different definitions of a positive result to be applied to the experimental data. The first definition, “singlets”, treated each reaction tube as an independent entity. The second definition, “duplicates”, required that both tubes containing the same reactants must be positive for the overall reaction to be considered positive. The best sensitivity and specificity for HS-LAMP singlets (96.7% and 92.5%) and duplicates (100% and 100%) was achieved at 27 and 28 minutes, respectively. The detection limit for template DNA amount was 5 pg. The sensitivity and specificity have not yet been determined using clinical specimens in the field. This thesis looked at HS in a funnel-shape pattern, from theoretical research to application. We first conducted a survey to understand the husbandry conditions in Pakistan and identify the variables that were statistically associated with the disease. Molecular epidemiology was then used to compare the genetics of different field and vaccine strains. Consequently, unique genes found only in HS-associated strains were identified. This allowed the design of a rapid and cheap diagnostic test suitable for the limited infrastructure in developing countries like Pakistan. Two important future directions of research are identifying potential virulence genes and validating the LAMP test under field conditions to evaluate its performance.

A molecular epidemiological study on Haemorrhagic septicaemia disease in Pakistan

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