Emergence of hemorrhagic septicemia caused by MLST type ST64 Pasteurella multocida in a European fallow deer population in Hungary

Abstract

A sudden mass mortality involving 44 fallow deer (Dama dama) occurred in September 2020 at a game management area near Budapest, Hungary. The die-off affected both sexes and all age classes equally. On postmortem examination of 2 subadults and 1 fawn, we found average body condition, diffuse subcutaneous edema, swelling of peripheral lymph nodes, marked hemorrhages in lymph nodes along the esophagus and trachea, and left ventricular dilation. We isolated Pasteurella-like colonies from spleen, liver, small intestine, lung, and brain samples of 7 animals, and identified them as P. multocida serotype B:2 by amplifying kmt1, toxA, and hyaC-hyaD genes, capsular typing by PCR, and serotyping by agar gel diffusion precipitation test, which established the diagnosis of hemorrhagic septicemia as the cause of the outbreak. By further analysis, we determined that all of our P. multocida isolates encoded ptfA, fimA, hsf-2, nanH, hgbA, and pfhA, but we did not detect genes encoding toxA, hsf-1, tbpA, or tadD. We identified our strains by RIRDC MLST as sequence type (ST)122, characteristic for strains causing hemorrhagic septicemia, while using multi-host MLST analysis we assigned these strains to ST64 clustering with P. multocida strains from the 2015 hemorrhagic septicemia outbreak of saiga antelopes in Kazakhstan. We concluded that increased population density and aggregation due to limited drinking water availability played a role in initiating the outbreak.

Keywords

fallow deer hemorrhagic septicemia

A large-scale sudden mortality of fallow deer (Dama dama) occurred in September 2020 at a hunting ground near Budapest, Hungary. In the 2-wk period between September 3 and 18, 43 fallow deer died with an additional carcass concluding the event on October 1. Both sexes were affected equally (23 females and 21 males), and the age distribution was 20 adults, 15 subadults, and 9 fawns. The site of the outbreak was an enclosed 600-ha hunting ground established in 2018 by fencing off a portion of a 2,000-ha estate. The outbreak site lies in a hilly area, covered by deciduous forest with enclosed patches of pine trees and game fodder fields. As the area had no springs or permanent water courses, the only source of drinking water was retained rainwater in natural and artificial water holes. Game management focused on maintaining a sustainable mixed species game population including a 95-strong fallow deer core population (as of 2020). At the time of the outbreak, other game species in the fenced area numbered 100–150 wild boar (Sus scrofa), 50 mouflon (Ovis musimon), and 30 red deer (Cervus elaphus). We estimated an event mortality rate of 40% in the total fallow deer population, comprising breeding-age adults and fawns.

Three fallow deer carcasses were submitted for postmortem examination on September 10. The 2 subadults and a fawn were in average body condition, weighing 32.2, 44.4, and 14.4 kg, respectively. Macroscopic findings included diffuse, moderate-to-pronounced subcutaneous edema, swelling of peripheral lymph nodes, and marked hemorrhages in the string of lymph nodes along the esophagus and trachea. We saw dilation of the left ventricles in all 3 animals, and confluent petechial epicardial and endocardial hemorrhages in the fawn. Moderate swelling and slight congestion were our only findings in the livers, kidneys, spleens, and lungs. Rumen contents of the fawn were foamy and watery with moderate amounts of fibrous feed remains. However, the 2 subadults and most of the animals autopsied in the field had dry, thick fibrous rumen contents, containing large amounts of acorn seeds and wild pear. Intestines were moderately autolytic with slightly swollen and hyperemic mucosa, distended blood vessels, and normal feces in the rectum of all animals. Mesenteric lymph nodes were swollen and had various degrees of autolysis.

We performed a routine bacteriologic examination of spleen, liver, small intestine, lung, and brain samples from each submitted carcass, along with 4 sets of organ samples (spleen, liver, and lung) collected from additional carcasses during on-site postmortem examinations. Samples were cultured at 37°C for 24 h on Columbia agar plates (Biolab) supplemented with 5% sheep blood. We isolated bacterial colonies exhibiting typical Pasteurella-like morphology from all organ samples. We identified 9 selected isolates from liver (3), spleen (2), brain (2), and small intestine (2; designated 4582–4590) as P. multocida using a multiplex PCR to amplify the species-specific kmt1, toxA (P. multocida toxin), and hyaC-hyaD (capsular type A) genes.^13,18 We identified capsular type other than type A and LPS genotype using multiplex PCR methods,^7,19 and we also carried out classical serotyping by the agar gel precipitation test.⁸ Testing of organ samples from the 3 fully examined cases by PCR for adenoviruses, bluetongue virus, epizootic hemorrhagic disease virus, and bovine viral diarrhea virus yielded negative results. Based on the above findings, we confirmed the diagnosis of hemorrhagic septicemia as the cause of the outbreak.

Hemorrhagic septicemia is an acute, frequently fatal disease primarily affecting cattle and water buffaloes in tropical regions of the world caused by certain serotypes of P. multocida, of which B:2 is the dominant serotype in Asia and E:2 in Africa.^4,14 Hemorrhagic septicemia cases have been reported from cattle, swine, and wild ruminants in temperate climatic regions of Europe with increasing frequency.^1,15 Sporadic cases and outbreaks of hemorrhagic septicemia in wildlife have been diagnosed in elephants (Elephas maximus) in Asia, American bison (Bison bison) and elk (Cervus elaphus) in North America, dromedary camels (Camelus dromedarius) in Sudan, fallow deer in the UK, Denmark, and Australia,^2,4 and African forest buffalo (Syncerus caffer nanus).⁴ Hemorrhagic septicemia has been found responsible for several large-scale mortality events among the highly endangered saiga antelope (Saiga tatarica) in Kazakhstan.¹¹ Mortalities with clinical signs and lesions resembling hemorrhagic septicemia in African elephants (Loxodonta africana) in Zimbabwe and Botswana were caused by Bisgaard taxon 45, a bacterial clade closely related to P. multocida.⁶ The presence of P. multocida serotype B:2 in Hungary was reported from an outbreak of P. multocida B:2–associated generalized pasteurellosis in backyard pigs,²⁰ and an isolated case of hemorrhagic septicemia in cattle was reported a few years later.¹³ The above-reported emergence of P. multocida B:2 as well as a mass mortality of fallow deer due to hemorrhagic septicemia in Germany¹² is the latest evidence of the silent circulation of this agent in Europe.

We examined all 9 P. multocida isolates originating from the outbreak to gain further insight into their relationship with P. multocida strains that caused hemorrhagic septicemia at a range of geographic locations worldwide. Using established methods,^5,17 we tested for virulence-associated genes, including types I and IV fimbrial subunits (fimA, ptfA), autotransporter adhesins (hsf-1, hsf-2), tight adherence protein D (tadD), filamentous hemagglutinin (pfhA), iron acquisition proteins (hgbA, hgbB, tbpA), and neuraminidase (nanH). We typed the isolates using the multi-host multilocus sequence typing (MLST) and Rural Industries Research and Development Corporation (RIRDC; AgriFutures Australia) MLST schemes as described earlier.^3,16 Following the purification and sequencing of all PCR products at Macrogen (Amsterdam, The Netherlands), we aligned and compared nucleotide sequences using Geneious Prime (v.2023.2.1; Dotmatics), and analyzed sequence data with PubMLST (https://pubmlst.org/). We submitted isolate 4586 (id: 1758) to the P. multocida MLST database¹⁰ (https://pubmlst.org/bigsdb?page=info&db=pubmlst_pmultocida_isolates&id=1758) and produced 2 Bayesian phylogenetic trees from a selection of concatenated RIRDC and multi-host MLST sequences in Geneious Prime using the GTR+G substitution model and default settings of the MrBayes 3.2.6 plugin.

All 9 P. multocida isolates originating from the outbreak encoded ptfA, fimA, hsf-2, nanH, hgbA, and pfhA, but we did not detect genes encoding toxA, hsf-1, tbpA, and tadD. We identified our strains by RIRDC MLST as sequence type (ST)122, characteristic for strains causing hemorrhagic septicemia. Using multi-host MLST analysis, we assigned the strains to ST64, a sequence type first described in 2017 from a case of bovine hemorrhagic septicemia in Hungary.¹³ We assume that the 2017 ST64 multi-host MLST strain is an unlikely source of the fallow deer outbreak, given the geographic distance and the length of time between the 2 events, but future full genome sequencing could provide a definitive answer to this question. Unfortunately, we could not determine the source of the fallow deer infection, and, in the absence of further cases, there are no clues pointing to a potential reservoir population. The affected game had no direct contact with domestic animals apart from dogs, which occasionally accompanied tourists passing through the area, and the site does not lie on an important avian migration route. Apart from a need for the monitoring of susceptible host populations, the potential role of carnivores and particularly wolves in the spread of hemorrhagic septicemia–causing P. multocida strains could be a hypothesis worth investigating, as indicated in an earlier study.¹²

By comparing the topologies of hemorrhagic septicemia–causing P. multocida multi-host and RIRDC MLST sequence types of the PubMLST database isolates on Bayesian phylogenetic trees, we found distinct congruence patterns in isolate assignment to ST sets (Fig. 1). With the exception of 2 isolates from horses (P-mult-5-KZ, P-mult-15-KZ) all multi-host MLST ST64 isolates are classified as RIRDC ST122. However, RIRDC ST122 comprises isolates assigned to a range of non-ST64 multi-host MLST types such as ST61, ST44, or ST194 (Fig. 1). Our analysis shows that the 2020 P. multocida isolates from fallow deer belong to multi-host MLST ST64, the same sequence type as the strains that caused the 2015 hemorrhagic septicemia–related mass mortalities of saiga antelopes in Kazakhstan, but the lack of RIRDC MLST data precludes further comparison.¹⁴ We found that, unlike the saiga strains,¹⁴ the genomes of the fallow deer isolates lack the tadD and hsf-1 coding genes.

Figure 1.

The evolutionary history of Pasteurella multocida strains from various disease presentations and outbreaks was inferred using Bayesian analysis of multi-host MLST and RIRDC MLST concatenated sequences downloaded from the P. multocida PubMLST database and the sequence of P. multocida strain 4586 (ST64) isolated from fallow deer in our study (highlighted in red). We constructed the phylogenetic tree in Geneious Prime 2023.2.1 using the GTR+G substitution model and default settings in the MrBayes 3.2.6 plugin. Supports for each node are given as Bayesian posterior probability values on each branch. Host origin, disease, serotype, and capsular type (where available) of the strains are from the P. multocida PubMLST database.

We assume that predisposing factors, including increased population density and associated social stress, played a significant role in triggering this hemorrhagic septicemia outbreak. The estimated spring populations of fallow deer numbered 103 animals in 2018, 94 in 2019, 95 in 2020, and 61 after the outbreak in 2021. In contrast to the 43 fallow deer harvested in 2018, hunters shot only 7 animals from the 94-strong core population during 2019, representing only 16% of the 2018 hunting bag. This fact suggests that the fallow deer population size at the time of the outbreak had actually been significantly higher than the 95 individuals officially reported for Spring 2020. At the end of the hot and dry summer of 2020, the availability of drinking water was limited to a couple of waterholes, causing additional concentration of the susceptible deer population and further increasing the effect of the pre-existing social stress. As a primary health management intervention during and following the hemorrhagic septicemia outbreak, game managers removed all carcasses and supplied ample drinking water at multiple locations across the area. This effort aimed to disperse the deer population and reduce both stress and contact rates among the animals. No further mortalities occurred on site beyond the described time period.

The low detectability of P. multocida B:2 in clinically healthy carrier hosts beyond hemorrhagic septicemia outbreak periods⁴ and the technical challenges of performing adequate surveys make it a daunting task to identify latently infected animals and reveal the epidemiologic factors and processes leading to sudden hemorrhagic septicemia outbreaks. However, the efficiency of disease risk assessment in both game management and conservation management depends on the range and quality of available background data.⁹ Identification of highly susceptible host species and the analysis of ecologic and epidemiologic features of P. multocida strains causing hemorrhagic septicemia contributes to this body of knowledge.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no dedicated financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Károly Erdélyi

Tibor Magyar

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