Tularemia affects animal welfare, human health, and the environment and is thus better approached from a one-health perspective27. Several studies in the Northern hemisphere28, and more recently in Australia15,16, have provided a vital research track in the epidemiology of this disease. In contrast, studies in Africa are too limited and scarce. The aim of this study was to investigate the presence of tularemia in wild leporids collected in Northern Algeria. These animals are highly susceptible to F. tularensis infection and considered sentinel hosts for surveillance of tularemia. The strategy we used to detect F. tularensis in leporids mainly used molecular, histological and immunohistochemical analyzes of tissues taken from animals found dead or hunted. To the best of our knowledge, detection of F. tularensis by PCR or culture has not been previously reported in wild leporidae in Algeria or other African countries.
Animal tissue samples were tested using three qPCR assays of variable sensitivity and specificity. The Type B-qPCR test targets a specific junction between ISFtu2 and a flanking 3′ region, which is considered specific for F. tularensis subsp. holarctica26, the only tularemia agent found in Europe and Asia. The Tul4-qPCR assay targets a simple copy gene encoding a surface protein, which can be found in the genome of all F. tularensis subspecies causing tularemia and that of the aquatic bacterium F. novicida. Because F. novicida has never been isolated from lagomorphs or other animal species, and very rarely from human29, a positive Tul4 qPCR for the studied tissue samples likely indicated the presence of F. tularensis DNA. The ISFtu2 qPCR is considered highly sensitive because multiple copies of this insertion sequence are found in the F. tularensis genome. However, it lacks specificity because ISFtu2 is also found in many other Francisella species25.
Two animals were considered “probable” tularemia cases because some of their samples were positive for the three qPCR tests. Ten animals were considered “possible” tularemia cases because their samples were positive for the ISFtu2 and Tul4 qPCRs but not the Type B qPCR. Finally 19 leporids were “uncertain” cases because only samples positive for the ISFtu2 qPCR were found. For the remaining 43 animals, all the tested samples were negative for the three qPCRs. Overall, we detected F. tularensis DNA-positive samples in 12/74 (16.21%) leporids, which strongly suggest that tularemia is present in the lagomorph population of the study area. The positive Type B qPCR tests in two animals suggested that F. tularensis subsp. holarctica could be the involved subspecies. We did not confirm these data by isolating F. tularensis from the studied leporids. However, the isolation of this pathogen from human or animal samples is tedious and has a low sensitivity13. Moreover, most of our samples were not appropriate for F. tularensis culture because of their long-term preservation in ethanol 70° or 10% formalin. Further study using fresh (non-fixed) tissue samples from dead leporids collected in the same study area is needed to definitively confirm the presence of tularemia in these animals and characterize the F. tularensis subspecies and genotypes involved.
Although PCR is usually more sensitive than culture for detecting F. tularensis, it also has some limitations. Firstly, the DNA extraction from organs preserved in ethanol for several months was difficult although easier for spleen than for liver samples. Some tissue samples could be lysed only after overnight incubation with proteinase K. Secondly, tissue samples contained PCR inhibitors as demonstrated by better DNA amplification from some samples after their dilution in PCR grade water. To reduce the effect of PCR inhibitors, organ samples with negative qPCR were retested using Bovine Serum Albumin (BSA) and the Real-time PCR system TaqMan (Applied Biosystems, Munich, Germany)30. Finally, DNA regions to be amplified were optimized to obtain high sensitivity and specificity of qPCR tests.
IHC detection of F. tularensis in formalin-fixed tissue can be helpful for tularemia diagnosis31,32. For one possible tularemia case, F. tularensis could be detected on immunohistochemical (IHC) examination of a liver sample using a specific anti-F. tularensis antibody. The intensity and localization of positive staining were comparable to those previously recorded for other animals32,33. IHC did not provide interpretable findings for four other tested specimens. Such negative results might be explained by an inhomogeneous distribution of infectious foci in the involved organs as well as a low bacterial inoculum in infected tissues. This has been previously demonstrated in tularemia granulomatous lesions in cell types like epithelial cells of the kidney, testis, and epididymis, hepatocytes, and bronchiolar epithelial cells31. Besides, IHC is a delicate technology whose results are highly dependent on the quality and fixation time of the organ tissues34. IHC analysis of dead animal tissues remains challenging, especially in case of tissue necrosis34.
In our limited case series we found a F. tularensis infection prevalence in leporids of 2.7% (2/74) for probable tularemia cases and 16.2% (12/74) when considering both probable and posible cases. We cannot make a guess about the prevalence of tularemia because our series is not representative of the general lagomorph population in the study area. In Germany, F. tularensis DNA was detected in 1.1% of European Brown hares and 2.4% of wild rabbits collected between 2009 and 201435. Higher infection rates were reported in the same country, including 11.8% (100/848 animals) in hares collcted in the North Rhine-Westphalia region36 and 30% (55/179) in brown hares collected between 2010 and 2016 in Baden-Wuerttemberg37. In Hungary, the prevalence of tularemia in hares was evaluated at 4.9–5.3%38. In Portugal, prevalences of 4.3% and 6.3% were reported in brown hares and wild rabbits, respectively39. However, the comparison of the reported tularemia prevalences in leporids is irrelevant because studies involved different animal species and geographic areas, and used different methods for F. tularensis detection.
Two possibilities could explain the lack of detection of tularemia in Algeria before this study. The first hypothesis is that this disease was not searched for in previous years, while it could have been present in this country for decades. The second hypothesis is that tularemia was recently imported in Algeria. Migratory birds may have been involved in the long-distance spread of F. tularensis40. These hosts can be infested by ectoparasites such as ticks which are the primary vectors of tularemia41,42. They can also spread the bacteria in the hydro-telluric environment through their secretions and feces18,43,44. An alternative possibility is that F. tularensis-infected animals (especially game animals) have been imported in Algeria from endemic countries. Whatever the mode of introduction of tularemia in Algeria, the dissemination of this disease over time might have been facilitated by the ability of F. tularensis to infect multiple hosts and its better survival in a cool environment45, which characterizes Northern Algeria climate. The emergence or re-emergence of tularemia in other countries has been related to climate change, human-mediated movement of infected animals, and wartime resulting in a significant rise of F. tularensis infections in the rodent populations39,46.
In our study, infected animals were collected throughout 4 years, although more frequently in autumn. Probable and possible tularemia cases were mainly collected during the hunting season (i.e., September, October, November, and December). Animals could not be collected in February because of heavy rains and in May and June because it corresponds to female leporids’ lactation period. In most endemic countries, tularemia cases are typically more frequent in late spring, the summer months, and early autumn37,47,48,49,50. Occasionally, fatal tularemia cases in hares have been predominantly reported during the cold season11,51. The climatic conditions can affect tularemia outbreaks in animals, depending on the reservoir involved and the predominant modes of infection52.
We detected tularemia more frequently in female than in male hares, and the reverse was true for wild rabbits. The prevalence of tularemia in male or female lagomorphs varies between studies. In Sweden, Morener et al.50 reported a tularemia case series only involving male hares. In the same country, Borg et al.50 observed an overrepresentation of females in the epizootic of 1967. They suggested that, compared to males, females had a higher risk of exposure to infected mosquitoes or were more vulnerable to tularemia because they were pregnant or had just given birth to a litter50. Tularemia was found in a few juveline leporids, which might be explained by a shorter exposure time to F. tularensis, a higher death rates due to higher susceptibility to F. tularensis infection or easier predation by their natural enemies, or more frequent hunting of adults compared to the juveniles53.
Tularemia is usually more frequently detected in leporids found dead than in hunted animals. As an example, a German study reported a higher prevalence of tularemia in hares found dead (2.9%) than in hunted ones (0.7%)35. In our study, most qPCR-positive animals were hunted. Our study might not be representative of the prevalence of tularemia in either population because most collected animals had been hunted.
The incubation period and clinical presentation of tularemia in leporids vary according to the species considered. Tularemia is typically an acute disease in mountain hares (Lepus timidus) in Scandinavia and has a chronic pattern in European brown hares (Lepus europaeus) in Central Europe50. The incubation time and clinical presentation of tularemia can be different in Cape hares (Lepus capensis). Wild rabbits are less sensitive to F. tularensis infection than hares31,39,54. An extended incubation period and chronic evolution of tularemia would facilitate the detection of F. tularensis in infected animals. In our study, a similar tularemia prevalence was found in the Cape hares and wild rabbits, which might reflect exposure to a same biotope area and environmental reservoirs of F. tularensis.
The pathological lesions of tularemiia in leporids can vary according to the F. tularensis strain involved, the mode and route of infection, and the susceptibility and immune status of the host32,50. In the European brown hares, granulomas with central necrosis have been reported in the lungs and kidneys and occasionally in the liver, spleen, bone marrow, and lymph nodes50. In contrast, only acute necrosis in the liver, spleen, bone marrow, and lymph nodes have been found in Lepus timudus hares in Sweden50. The lesions in the Japanese hare (Lepus brachyurus angustidens) are comparable to those of Lepus timidus, except for cutaneous, lung, brain, and adrenal gland lesions32. In the European rabbit, Oryctolagus cuniculus, tularemia is not associated with identifiable macroscopic tissue lesions39,55. To our knowledge, no reports describing post-mortem lesions in Cape hares with tularemia are available. In this study, similar lesions were found in hares and wild rabbits except necrotic foci only observed in some wild rabbit organs (such as liver, lungs, kidney, ovary). Most animals had pathological lesions of pneumonia, gastritis and enteritis. Kidney lesions and adrenal glands enlargment were oberved. Necrotic lesions were occasionally found in the lungs, liver, spleen and ovary and hemorrhages in the lungs, liver, and intestines.
Tularemia is an arthropod-born disease in most endemic areas14,22,28. In our study, 50% of positive leporids were infested by known tularemia vectors such as ticks (Ixodes ricinus56,57, Rhipicephalus sanguineus39), fleas (Spillopsylus cuniculi58), and lice of lagomorphs (Haemodipsus lepori and Haemodipsus setoni59,60). Ticks are the most significant arthropod vectors of tularemia61. Ticks are frequently involved in the transmission of tularemia in North America, including Dermacentor andersoni, D. variabilis, and Amblyomma americanum57,62,63. In Europe, tick-borne tularemia represents 13% to 26% of human cases57,64. The involved species include D. marginatus, D. reticulatus, I. ricinus, R. sanguineus, and Haemaphysalis concinna65,66. Further research on wild leporid sucking arthropods is needed to confirm the presence and clarify the ecology of F. tularensis in Algeria.
Our study reports for the first time the detection of F. tularensis DNA in leporids from Northern Algeria. The markers most in favor of tularemia in the animals studied are the positivity of qPCR tests, in particular, the “type B” qPCR test which amplifies a specific DNA sequence of F. tularensis subsp. holarctica, and a positive immunohistological examination in one animal. Further investigation is needed to confirm our results by the isolation of this pathogen from animal samples and determine the F. tularensis subspecies and genotypes involved. This would allow the characterization of the F. tularensis subspecies and genotypes present in Algeria. Furthermore, our findings push us in future studies to seek tularemia in the Algerian human population. To achieve this, interdisciplinary or trans-disciplinary collaborative efforts underpinned by the One Health concept will be necessary.
Source: Ecology - nature.com
