Abstract
Trypanosoma evansi is a protozoan blood parasite and etiologic agent of “surra,” a disease affecting a wide range of domestic and wild mammals, some identified as potential reservoirs. Although T. evansi has been detected in several small wild rodent species, their role in the epidemiology of surra is unclear. There is molecular evidence of T. evansi in wild rodents in Asia, but it is not known whether they can carry the parasite for sufficient time to significantly contribute to the epidemiology of surra. We assessed the susceptibility of the Oriental house rat (OHR; Rattus tanezumi) to T. evansi infection. Five adult male OHRs trapped in Bangkhen district, Bangkok, Thailand, and five laboratory Wistar rats (Rattus norvegicus) as positive controls, were experimentally infected with a local strain of T. evansi. The five controls and three of the five OHRs were highly susceptible and rapidly exhibited the high levels of parasitemia usually observed in Wistar rats. They died or were euthanized just prior to expected death. Two OHRs presented fluctuating levels of parasitemia, without obvious clinical signs, throughout 40 d of monitoring. These results highlight the moderate susceptibility of some OHRs and their ability to carry the infection over time. Along with the molecular evidence of T. evansi in captured OHRs (demonstrated elsewhere), our results bring new information on the potential role of OHRs in the complex epidemiology of surra.
Trypanosoma evansi (Kinetoplastida, Trypanosomatidae) is a protozoan blood parasite that causes “surra” in domestic and wild mammals. As the most-widely distributed pathogenic mammal trypanosome (Hoare 1972), T. evansi has been found in numerous mammalian hosts among which the clinical expression differs, many of them being asymptomatic and potential reservoirs (Desquesnes et al. 2013b).
Few studies have explored the role of small wild rodents in the epidemiology of T. evansi. The parasite was detected using the hematocrit centrifuge technique (HCT), including direct observation of the parasite in the buffy coat (Woo 1970) in three rodent species in Brazil (Rademaker et al. 2009). In Thailand, T. evansi was detected in blood samples by PCR in six murine species (Murinae) including the Oriental house rat (OHR; Rattus tanezumi) (Milocco et al. 2013; Pumhom et al. 2013). The OHR is widespread and abundant in Asia, including all of Thailand, and is found in diverse habitats including urban, agricultural, and forest areas (Heaney and Molur 2008). The OHR are likely to live near various known domestic and wild hosts for T. evansi and could, therefore, have a significant impact on its transmission. However, because these studies involved samples from one time point, they do not provide evidence for whether the OHR can sustain T. evansi infection long enough to contribute to its transmission. To evaluate this potential, we experimentally assessed the susceptibility of the OHR to T. evansi.
Five adult male OHRs were trapped on the Kasetsart University Bangkhen campus, Bangkhen District, Bangkok, Thailand and identified using morphologic criteria available on the CERoPath website (Centre de Coopération Internationale en Recherche Agronomique pour le Développement 2014). Five adult male Wistar rats (Rattus norvegicus) were used as positive controls. Rats were kept individually in cages under standard conditions for laboratory rodents (Wolfensohn and Lloyd 2013).
To ensure that the rats had not been infected with trypanosomes, 70 μL of blood were collected in a heparinized capillary tube by saphenous venipuncture and examined for trypanosomes using HCT (centrifugation of the capillary tube and microscopic examination of the upper layer of the buffy coat; Woo 1970). Plasma was used to perform an indirect enzyme-linked immunosorbent assay (ELISA) using soluble T. evansi antigens and peroxidase-conjugated anti-rat immunoglobulin G (A9037, Sigma-Aldrich, St. Louis, Missouri, USA), following Desquesnes et al. (2009). The DNA was extracted from packed cells and T. evansi and Trypanosoma lewisi DNA was detected by PCR using TEPAN and LEW primers, respectively (Pruvot et al. 2010; Desquesnes et al. 2011).
To generate parasites for experimental infections, a Wistar rat (WR120F) was inoculated intraperitoneally with cryopreserved T. evansi isolated in 2011 from a naturally infected horse in Ratchaburi province. After 5 d, the rat was anesthetized using ketamine (intramuscular; 20 mg/kg) and euthanized to collect blood. Parasitemia (number of trypanosomes/mL blood) was estimated following Herbert and Lumsden (1976), and 100 μL of blood diluted in phosphate-saline-glucose buffer, containing approximately 5,000 trypanosomes, was injected subcutaneously into the five OHRs and five Wistar rats.
After infection, a drop of blood from the tip of the tail was examined daily at 400×. The mean number of trypanosomes in 20 microscope fields was counted to estimate parasitemia. When no parasites were detected for 4 d, blood was collected in a capillary tube as described above and submitted to HCT. If parasites were not found by HCT, PCR was performed to detect T. evansi DNA using TEPAN primers. This protocol was designed to regularly detect low quantities of parasites with limited stress to the rats. Although HCT and PCR are more sensitive than direct blood examination, collecting blood in capillary tubes requires stressful manipulation of the animals that might interfere with immune control of infection. On day 40, the surviving rats were anesthetized and euthanized by exsanguination.
All rats were negative by ELISA for T. evansi and by PCR for T. evansi and T. lewisi DNA prior to experimental infection. After parasite injection, the five positive controls presented rapidly increasing parasitemia detectable by direct blood examination. All died within 10 d of inoculation, with parasitemia levels above 50 million trypanosomes/mL of blood on the day before death. Three of five OHRs presented similar parasitemic profiles to those of the Wistar rats, rapidly leading to high levels of parasitemia and death within 8 d of inoculation (Fig. 1). The two remaining OHRs presented irregular parasitemia detectable by direct blood examination, HCT, or PCR throughout the experiment. On day 40, no parasites were detected by direct blood examination in these two rats and they showed no clinical signs before euthanization.
Monitoring of five Oriental house rats (Rattus tanezumi), Rt1–Rt5, experimentally infected with Trypanosoma evansi. Parasitemia levels estimated by counting on wet blood films are given on a semiquantitative scale (left axis). For Rt4 and Rt5, results of the enrichment method are presented on the same chart.
Monitoring of five Oriental house rats (Rattus tanezumi), Rt1–Rt5, experimentally infected with Trypanosoma evansi. Parasitemia levels estimated by counting on wet blood films are given on a semiquantitative scale (left axis). For Rt4 and Rt5, results of the enrichment method are presented on the same chart.
That all individuals developed detectable parasitemia after inoculation reveals the susceptibility of R. tanezumi to T. evansi. Survival for >40 d without clinical signs, as we observed in two OHRs, is not observed in laboratory rats. More than 30 Wistar rats have been infected for other purposes in our laboratory and all progressed quickly toward death. The variable response to infection in R. tanezumi may be due to intraspecific genetic diversity among wild individuals while laboratory rats have a uniform genetic background. This is speculative, however, because few individuals were included in the experiment and other factors such as age or concomitant infections could have interfered. Whatever the explanation, the moderate susceptibility of some OHRs raises the question of their potential role in the epidemiology of surra, possibly as a reservoir.
Additional questions remain unanswered, including possible modes of transmission of T. evansi in wild rodents. In large herbivores, mechanical transmission by biting insects such as tabanids (Tabanidae) or stomoxes (Stomoxys spp.) is considered the primary route of infection (Baldacchino et al. 2013, 2014). However, these insects are diurnal and are attracted by large animals (Foil et al. 1994) while wild rodents are small and mostly nocturnal. Other potential vectors could be investigated such as fleas (Siphonaptera; cyclical vectors of Trypanosoma lewisi) and sand flies (Phlebotominae). Oral transmission of T. evansi by ingestion of contaminated blood, as described in dogs (Desquesnes 2004), was experimentally demonstrated in mice (Mus musculus; Raina et al. 1985) and Wistar rats (Vergne et al. 2011). Under natural conditions, OHRs may become infected through ingestion of blood, fresh dead animals, or peripartum tissues. Indeed, OHRs are omnivorous and may feed on carrion as does the black rat (Rattus rattus; Burnie 2001). Direct trypanosome transmission between conspecific rodents through biting has also been suggested (Smith et al. 2006). Transmission of the parasite to susceptible predators or scavengers, such as dogs or pigs, may also be possible orally through consumption of infected rodents (Desquesnes et al. 2013a). Transmission from rodents to herbivores seems less likely, even though mechanisms could be suggested such as accidental via contaminated blood in ticks that fed on rats (Vergne et al. 2011).
Further investigations are required to explore these hypotheses. Similar experiments lasting >40 d should be conducted with individuals infected orally, as is likely to occur in natural conditions and might increase the rate of subclinical infections. Experiments with other wild rodent species in which T. evansi has been detected should also be conducted.
Although T. evansi was detected in various species of rodents under natural conditions, there is no evidence that they are involved in surra outbreaks or in the sustainability of T. evansi circulation (e.g., Rodríguez et al. 2010). Our study brings new information in this regard, but other laboratory and field studies are needed to fully explore the potential role of OHRs in the epidemiology of surra.
We are very thankful to Peter Biggins for revision of the English manuscript.