This dynamic can be observed from the 2007 RVF outbreak in Gezira State, Sudan (**Figure 5**), when satellite monitoring (June– September, 2007) showed that most of the central Sudan could be unusually subjected to heavy rainfall (76). Accordingly, a RVF risk warning has been generated for central and southern Sudan. Indeed, the predicted unusually heavy rains occurred during July– August and resulted in severe floods (77). In September, suspected human RVF cases were reported (78). The first cases appeared in southern areas of Algabalain locality in White Nile state. The first symptoms among the suspected cases were hemorrhage and fever with rapid death. All reported cases in the beginning of the outbreak were scattered and did not reach any health facilities (21). First human index case was confirmed on 8–14 October, 2007 (76). The RVF outbreak in Sudan came to public attention on October 18,2007 when the FederalMinistry of Health (FMoH) Sudan asked the WHO to assist in the investigation and control of a suspected hemorrhagic fever. The WHO and FMoH teams started investigations in the White Nile state, central Sudan, on October 24, and on the basis of initial results, an outbreak of RVF was declared on October 28, and more help was requested for control measures (79). An announcement was made regarding RVF in animals (80) when the outbreak reached its peak in humans by early November (**Figure 5**). At the end of the outbreak as of late December 2007

to January 2008, a cumulative total of 698 cases, including 222 deaths, was reported from six states (Gezira, Kassala, Khartoum, River Nile, Sennar, and White Nile), yielding an overall CFR of 31.8%. This RVF outbreak was the first one reported in humans and connected directly to heavy rainfall, flooding, and increase in mosquito breeding sites in Sudan (81).

The RVF outbreak of 2007 in Sudan not only validated the association between abnormal rainfall and RVF outbreak but also prediction of RVF outbreak and early warning signs from satellite monitoring. This also showed that the wave of RVF outbreak is likely to end as the water pools due to rainfall and warm temperatures faded-out (68). This is indeed the case as there is only one short-rainy season in Sudan, which ends in October and then the winter season begins at the end of November and runs up to late February. The presumed link between extraordinary flooding events and RVF outbreaks was also well validated, among others, by a successful prediction of the 2007 outbreak in Somalia,Kenya, and northern Tanzania, using climate modeling (82). In fact, each of the seven documented moderate or large RVF outbreaks that have occurred in East Africa over the last 60 years have been associated with El Niño Southern Oscillation (ENSO) associated with above normal and widespread rainfall (83). This association of RVF with

and temporal window sizes and that such cluster could include a maximum of 50% of all cases. They indicated there were no clusters detected in 1930, from 1947 to 1978 three primary clusters were persistently detected in

cluster is displayed (RR) along with the buffer (circle) size in kilometers (km). Source: Sindato et al. (68), with permission from Calvin Sindato, National Institute for Medical Research, Tabora, Tanzania.

excessive rainfall and flooding was also observed in other countries outside the African continent in Arabian Peninsula, i.e., the outbreak of 2000 in Yemen (84).

Interestingly, all RVF outbreaks in Sudan originated in White Nile State where the first RVFV was identified in 1973 as the cause of an extensive epizootic (10), then moved northward through the White Nile river valley to Khartoum in 1976 (85) and extended to the neighboring states of Gezira, Sennar, and Kassla states during 2007 outbreak (**Figure 1**). In theWhite Nile State, the river Nile has a very wide basin, which floods annually between June and September, resulting in wide wetland along the valley of the river from the border of Southern Sudan up to Khartoum. It is important to mention that the five states are located in the Central Clay Plain soil of the Sudan, which extends from west of Kassala through Gezira, Khartoum,White Nile up to southern Kurdufan. This type of soil and topography when flooded create large shallow wetlands similar to what is known as "*dambo*," which is often shown as suitable breeding habitats for *Aedes* mosquitoes in central, southern, and eastern Africa. This suggested that local environment is very important and is directly linked to RVF outbreak. Significant association was observed between RVF outbreaks from 1930 to 2007 and clay and loam soil textures in the eastern Rift Valley ecosystem of Tanzania where clustering of RVF outbreaks were persistently and predominantly detected (68). Clay soil rather than sandy soil texture supports long-period retention of water contributing to

flooding and wetness of habitat suitable for breeding and survival of *Aedes* mosquito vectors. This suggests that while rainfall might be the major determinant for the onset and switch-off of an outbreak, it is unlikely that it is the only factor responsible for the spread and clustering of RVF cases. A causal association between local environmental factors, livestock density and movement, encroachment of mosquitoes into new areas, and occurrence of RVF has been suggested in previous studies (20, 86, 87).

## **RISK FACTORS DURING RVF OUTBREAK**

It is generally accepted that during the 2007 outbreak in Sudan, animal contact was the most dominant risk factor followed by animal products and mosquito bites (78). This is supported by the fact that the 2010 outbreak was first characterized by abortions in ewes followed by infections in persons with histories of contact with aborted fetal material (8). Contact with RVFV-infected animals such as consuming or handling products from sick animals, touching an aborted animal fetus, or being a herdsperson has been documented as the most important risk factor for severe infection during the 2007 outbreak in Kenya (76). A similar result was observed during the previous RVF outbreak of 1997–1998 in northern Kenya (11). These findings are consistent with thosefrom another study from Sudan stating that most of the animals such as sheep, cattle, goats, and camels stay very close to their owners' houses at night (21, 88).

such cluster could include a maximum of 50% of all cases. They conducted the analysis of clustering of cases separately for humans and domestic ruminants during the 2006/2007 outbreak wave. Between

each cluster is displayed (RR) along with the buffer (circle) size in kilometers (km). Source: Sindato et al. (68), with permission from Calvin Sindato, National Institute for Medical Research, Tabora, Tanzania.

Despite the fact that there was no evidence for horizontal transmission between humans in Sudan or elsewhere, risk from infected pregnant women through vertical transmission can occur. During the 2007 outbreak in Sudan, a 29-year-old pregnant woman presented in early labor with symptoms suggestive of RVF and delivered a baby weighing 3.2 kg with skin rash, palpable liver, and spleen. Two samples from the mother and neonate were screened and found to be positive for RVF-IgM (89). This case demonstrated that RVF can be vertically transmitted in human. A similar case was also reported before in Saudi Arabia, during the 2000 outbreak (90). These are consistent with the claims recently made about the burden of emerging zoonotic infectious disease among women in general and pregnant women, in particular (91).

Movement of animals during an outbreak can be a serious risk factor. Complete genome sequences from RVFV strains detected during the 2007 and 2010 outbreaks in Sudan suggested multiple introductions of RVFV into Sudan as part of sweeping epizootics from eastern Africa (8). All RVFV strains observed grouped into Kenya-1 or Kenya-2 sub lineages, which defined the eastern Africa outbreak in 2006–2008 (92). The sequencing also suggested that an earlier common ancestor from 1996 coinciding with the 1997–1998 outbreaks in the horn of Africa. The Kenya-2 sub lineage is now known to be widely distributed in Tanzania and Sudan (8, 92, 93). The movement of animals from southern states in White Nile to northern ones in Gezira, Khartoum, and Kassala for marketing was most likely responsible for the geographical expansion of the virus in central and eastern Sudan (**Figure 2**). Identical or nearly identical sequences of the virus strains were identified for different states and years, Khartoum in 2007 and Gezira in 2010, as well as Khartoum and West Nile in 2007. These sequences indicate recent movement of the virus in this region and support the necessity and utility of surveillance systems for recognizing when and where a large epidemic is imminent (8).

## **SURVEILLANCE AND CONTROL OF RVF OUTBREAK: THE EXAMPLE OF SAUDI ARABIA IN 2000**

Saudi Arabia and Yemen experienced a huge RVF outbreak in the year 2000 (29, 30, 54, 55). It was the first outbreak in Middle East outside its endemic areas in Africa. The outbreak in Saudi Arabia is suggested to have been from eastern Africa by importation of infected animals (40), similar to the suggested route of introduction of RVFV into Egypt in 1977 from Sudan (94). The virus causing the Saudi Arabia outbreak belonged to the same strain that caused the 1997–1998 outbreaks in East Africa (95).

After the outbreak was declared, a team was established in collaboration between the Ministries of Health, Agriculture, and Water, and the Ministry of Municipalities and international organizations such as CDC, WHO, and National Institute of Virology, South Africa, to control the outbreak (30, 54, 96). A strategy called "One Health" was then implemented by Saudi Arabia targeting

both the animal and human hosts (17). The urgent integrated control measures that were implemented by this strategy during the outbreak included the following activities: (1) disposal of dead animals in an appropriate manner, (2) active surveillance surveys to detect cases of RVF among humans and animals to locate target areas for animal vaccination, and (3) apply a vaccination campaign that started in October 2000 (16, 30, 31, 54, 96). Around 1,200,000 doses of the vaccine were reported to be imported into Saudi Arabia and the campaign continued in 2001 with more than 10 million ruminants being vaccinated (31). These activities were accompanied by (4) a restriction on animal movements outside the affected areas and a ban on animal imports from RVF-enzootic countries (29). (5) For effective case management, detailed case definition was developed, training sessions on how to manage the suspected cases clinically was implemented, two well-prepared laboratories (one in the affected regions and the other in the capital of the country) for diagnosis of RVFV antibodies in suspected cases were also provided by the Saudi Ministry of Health (29, 54, 96). (6) Epidemiological investigation was also performed to identify risk factors (30). (7) In addition, an entomological study to search for the mosquito breeding grounds (30) was followed by an intensive mosquito control program with spraying (54, 97). This strategy succeeded to limit the effect of the outbreak and curb the disease from spreading to other areas. Since 2000, only sporadic cases have been recorded in Saudi Arabia and only in the same regions where the original outbreak was reported (16). Later investigation on this strategy concluded that "One Health" approach is the best option to mitigate outbreaks of RVF. Collaboration between veterinary, health, and environmental authorities both at national and regional levels is needed to control RVF outbreak (17).

## **CONCLUSION AND PERSPECTIVE**

From the foregoing narrative, we can conclude that RVF causes huge health and economic losses signified by the number of human Himeidan et al. Insights into recent RVF outbreaks

deaths and high mortality and abortion rates in livestock. It is also clear that whereas RVF was previously restricted to specific areas in sub-Saharan Africa, the disease seems to be spreading into new territories beyond the traditional foci as evidenced by outbreaks in the Arabian Peninsula. The epidemiology of RVF is complex and transmission involves multiple mosquito vector species. A multiplicity of factors shapes the epidemiology of RVF. Key among these is rainfall and flooding, soil types, contact with animals, breeding sites, and availability and movement of livestock. Epizootics are interspaced with long periods of quiescence.

It is our considered view that repeated outbreaks could be forestalled with adequate sensitization of the policy makers. It is also clear that with enhanced coordination among stakeholders, e.g., Ministries of Health and Livestock, researchers, and local communities it is possible to better handle future outbreaks. Such coordination of stakeholders seems to have worked effectively in managing the outbreak in Saudi Arabia. Other regions such as eastern Africa that has borne the brunt of previous outbreaks should learn from the Saudi experience. In light of improved warning signs derived from satellite imagery and mapping, governments should come up with clear strategies and action plans for preparedness and handling of future outbreaks. Such strategies should include strong surveillance systems, adequate and well trained personnel, among others.

## **AUTHOR CONTRIBUTIONS**

Yousif E. Himeidan suggested the topic, framed, drafted, and wrote up the manuscript. Mostafa M. Mahgoub collected the data on patients from Ministry of Health, Gezira State. Eliningaya J. Kweka, El Amin El Rayah, and Johnson O. Ouma drafted and reviewed the manuscript. All authors read and approved the final version.

## **ACKNOWLEDGMENTS**

We thank the staff in the Epidemiology Unit, Ministry of Health, Gezira State for providing the data on patients of the 2007 outbreak.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 29 July 2014; accepted: 16 September 2014; published online: 06 October 2014.*

*Citation: Himeidan YE, Kweka EJ, Mahgoub MM, El Rayah EA and Ouma JO (2014) Recent outbreaks of Rift Valley fever in East Africa and the Middle East. Front. Public Health 2:169. doi: 10.3389/fpubh.2014.00169*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Himeidan, Kweka, Mahgoub, El Rayah and Ouma. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Comparative study of the pathological effects of western equine encephalomyelitis virus in four strains of Culex tarsalis Coquillett (Diptera: Culicidae)

#### **Marco V. Neira1,2\*, Farida Mahmood3,4,William K. Reisen4 , Calvin B. L. James 2 andWilliam S. Romoser 2**

1 Center for Infectious Disease Research, College of Exact and Natural Sciences, Pontificia Universidad Católica del Ecuador, Quito, Ecuador

2 Department of Biomedical Sciences, Tropical Disease Institute, College of Osteopathic Medicine, Ohio University, Athens, OH, USA

3 Environmental Health and Engineering, United States Army Public Health Command Region-South, Houston, TX, USA

4 Center for Vector-borne Diseases, School of Veterinary Medicine, University of California, Davis, CA, USA

#### **Edited by:**

Juan-Carlos Navarro, Universidad Central de Venezuela, Venezuela

#### **Reviewed by:**

Jia Liu, Pfizer Inc., USA Maria Julia Dantur Juri, CONICET, Argentina

#### **\*Correspondence:**

Marco V. Neira, Center for Infectious Disease Research, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Avenida 12 de Octubre 1076 y Roca, Quito, Ecuador e-mail<EMAIL_ADDRESS>
Early reports suggested that mosquito cells infected with arboviruses remain viable and undamaged. However, more recent experimental evidence suggests that arboviral infection of mosquito tissues might indeed result in pathological changes, with potential implications for vector survival and virus transmission. Here, we compare the pathological effects of western equine encephalomyelitis virus (WEEV) infection in four strains of Culex tarsalis previously reported to differ in their competence as WEEV vectors. Pathological effects were observed in cells of the midgut epithelium, salivary glands, and eggs. Cell rounding and sloughing of midgut epithelial cells was associated with those strains reported to be the least susceptible to WEEV infection, whereas midgut necrosis and vacuolation upon infection were associated with strains showing higher susceptibility. Although pathological effects were sporadically observed in infected salivary glands, further studies are required to evaluate their impact on vector competence. Additionally, the potential implications of observed C. tarsalis egg infection with WEEV are discussed.

**Keywords: arbovirus, Culex tarsalis, mosquito, pathology, vector competence, western equine encephalomyelitis**

## **INTRODUCTION**

Transmission of a mosquito-borne virus to a vertebrate host requires mosquito ingestion of a viremic blood meal, subsequent infection of the mosquito's midgut cells, spread to tissues within the hemocoel, and finally infection of the salivary glands. Following completion of this "extrinsic incubation period," virions must be released into the saliva and injected into a new host during a subsequent blood meal. Previous investigations have described benign, non-pathological, and chronic viral infections resulting in continuous virus production throughout the lives of infected mosquitoes (1–3). However, pathological effects have also been observed both *in vivo* (4–10) and *in vitro* (11). Weaver et al. (6) reported pathological changes, including cell sloughing and tissue necrosis in *Culex tarsalis* that fed on viremic blood meals containing western equine encephalomyelitis virus (WEEV; Togaviridae, *Alphavirus*). Furthermore, it has been proposed that the effects exerted by viral infection in the mosquito can influence vectorial capacity (12).

The temporal dynamics of WEEV infection and associated variations in transmission have been described for four strains of *C. tarsalis* that differed in their susceptibility to WEEV (13, 14). Here, we describe varying tissue pathology associated with WEEV infection in these four strains and discuss the potential influence of these variations on vector competence.

## **MATERIALS AND METHODS**

#### **MOSQUITO REARING AND INFECTION**

Mosquito rearing and handling methods used in this study have been previously described (13). Briefly, larvae were reared at 22–24°C, with a 16 h light:8 h darkness photoperiod, and were fed ground alfalfa pellets and AquaMax® (Purina Mills, LLC; St. Louis, MO, USA). Adults were maintained under a similar photoperiod at 26°C and were provided a 10% sucrose solution *ad libitum*.

Four strains of *C. tarsalis* were used in the current study: (a) WEEV resistant (WR), (b) high viremia producer (HVP), (c) Coachella Valley (COAV), and (d) Kern National Wildlife Refuge (KNWR). The WR and HVP strains were selected for refractoriness or high susceptibility, respectively, to infection with WEEV (the HVP strain was derived from the original WEEV susceptible –WS- strain) (15, 16) and have been maintained at the University of California Arbovirus Field Station since the mid 1980s. In preparation for this study, the HVP and WR strains were reselected for several generations by examining the susceptibility of single families (13). The COAV and KNWR strains were collected in California's Riverside and Kern counties, respectively, and had been maintained as unselected colonies for 2 years prior to this study.

For viral infections, we used the WEEV strain BFS1703, which was isolated from *C. tarsalis* collected in Kern County, California, in July 1953 (17) and has been widely used for evaluating the competence of *C. tarsalis* to transmit WEEV (15, 18, 19). Virus was passaged twice in suckling mice and once in Vero cell culture prior to the beginning of the study.

The method used to infect mosquitoes has been previously described (13). Briefly, three to five day-old mated females were starved for 18 h and then allowed to engorge on viremic blood via an artificial membrane feeder (13, 20). Blood solutions contained *ca*. 3 or 5 log10 plaque forming units (PFU) of WEEV per 0.1 ml of chicken blood containing 14.3 freeze dried USP units of sodium heparin per milliliter (Becton-Dickson, Franklin Lakes, NJ, USA). Hereafter, these viral doses will be designated as '3-log' and '5-log'. The 5-log dose was comparable to viremias produced by competent avian hosts that were able to infect most competent vectors, whereas the 3-log dose was similar to that produced by a less competent host, but still able to infect highly susceptible mosquito hosts such as the HVP strain. Doses below this were insufficient to infect most mosquitoes (21).

For uninfected controls, mosquitoes were allowed to feed on virus-free blood by the same method. Fully engorged females were transferred to an incubator maintained at 26°C and 18 h light:6 h darkness photoperiod, and provided with 10% sucrose solution that was changed daily.

### **IMMUNOCYTOCHEMISTRY**

For each strain, two uninfected controls and five individuals fed on each viral dose were collected at days 1, 2, 3, 4, 7, 14, and 21 post-infectious blood meal (DPI). These mosquitoes were immobilized on wet ice, killed and fixed by injection of 10% buffered formalin (pH 7.5), and stored in 100% ETOH until further processed. Subsequently, these specimens were dehydrated, cleared, and infiltrated with paraffin as previously described (22, 23), embedded in paraffin blocks, cut into 10µm thick serial longitudinal sections using an American Optical® 820 Spencer™ microtome (American Optical Co., New York, NY, USA), mounted on microscope slides, and stored at 4°C until used for immuno-staining.

Mounted sections were immuno-stained as previously described (14). Briefly, the avidin–biotin-peroxidase complex (ABC) technique was applied, using a 1/1,600 dilution of mouse anti-WEEV ascites fluid as the primary antibody, and the horse-anti-mouse Vectastain Elite® ABC kit (Vector laboratories, Burlingame, CA, USA) for detection, following the manufacturer's protocols. Stained sections were examined for the presence of viral antigens – as evidenced by the rustybrown color generated by the ABC technique (**Figure 1**) (22)– as well as for the presence of any pathological changes, using a Nikon® Optiphot™ compound microscope (Nikon

Instruments Inc., Melville, NY, USA) equipped with a digital Spot RT™ camera (Diagnostic Instruments, Sterling Heights, MI, USA).

#### **DATA ANALYSIS**

Statistical analyses were performed using the SPSS® software package version 13.0 for windows (SPSS Inc; Chicago, IL, USA). Chi-Square tests were used when comparing overall frequencies (i.e., time groups pooled together) between 3- and 5-log groups in each strain. If no significant differences (*P* > 0.05) between the dose groups were found within a strain, dose groups were pooled for further analysis; otherwise, each dose group was analyzed separately.

Analyses of differences among strains were performed using Kruskal–Wallis (K–W) tests because frequency data were not normally distributed for at least one strain in each one of the parameters studied. If K–W tests indicated significant (*P* < 0.05) differences among strains, *post hoc* analysis was performed by applying Chi-Square tests to all pair-wise combinations of strains. To maintain an overall alpha level of 0.05, a Bonferroni correction was applied to the *post hoc* testing.

## **RESULTS**

Pathological changes in infected individuals were consistently observed in the midgut epithelium (**Figures 1**–**3**) and eggs (**Figure 4**). Additionally, atypical cellular morphology was sporadically observed in the salivary glands of infected individuals (**Figure 5**); however, the consistency of salivary acini often caused them to be detached from the slides during the washes required for immunocytochemical staining, making it impossible for us to obtain consistent data across all experimental groups for this particular tissue. Therefore, only the pathological changes observed in the midgut and eggs will be further reported and discussed.

#### **MIDGUT PATHOLOGY**

Three types of pathological changes were found in infected midguts: vacuolation, necrosis, and cell rounding and sloughing (CRS, **Table 1** and **Figure 6**).

**FIGURE 1 | Vacuolation of midgut epithelium**. **(A)** Section of the posterior midgut of a specimen of the COAV strain, 3 days after ingesting a blood meal containing 5-log PFU of WEEV per 0.1 ml blood. Notice the extensive formation of vacuoles in the cytoplasm. Rusty-brown staining is indicative of a positive immunocytochemical reaction, denoting the presence of WEEV antigen in the tissue. **(B)** Comparable section of the posterior midgut in an uninfected control.

**FIGURE 2 | Midgut epithelium necrosis**. **(A)** Section of the posterior midgut of a specimen of the HVP strain, 2 days after ingesting a blood meal containing 5-log PFU of WEEV per 0.1 ml blood. Notice how the epithelium has become necrotic, being reduced to a very thin band, with neither

discernible cell boundaries nor traces of cytoplasm or organelles. **(B)** Comparable section of posterior midgut in an uninfected control, where no necrosis is observed. BM, blood meal (in the midgut lumen); ME, midgut epithelium; NE, non-infected egg.

**FIGURE 3 | Cell rounding and sloughing (CRS)**. **(A)** Anterior midgut section of a specimen of the KNWR strain, 14 days after ingesting a blood meal containing 3-log PFU of WEEV per 0.1 ml blood. Notice how several epithelial cells have sloughed-off into the lumen, and some rounded cells protrude from the tissue. **(B)** Comparable section of anterior midgut in an uninfected control, where no CRS is observed. FB, Fat body; L, midgut lumen; M, skeletal muscle; ME, midgut epithelium; R, rounded epithelial cell; S, sloughed epithelial cell.

**FIGURE 4 | Egg infection and pathology**. **(A)** Longitudinal section of the abdomen of specimen of the COAV strain, 21 days after ingesting a blood meal containing 5-log PFU of WEEV per 0.1 ml blood. Rusty-brown staining is indicative of a positive immunocytochemical reaction, denoting the presence of WEEV antigen in the tissue. Notice the smooth yolk

texture observed in infected egg, in contrast with the uniformly granular texture of yolk observed in the neighboring uninfected eggs. **(B)** Comparable section of the abdomen of a specimen of the COAV strain, showing only normal, uninfected eggs. IE, infected egg; UE, uninfected egg.

**FIGURE 5 | Pathology in salivary glands**. **(A)** Salivary gland of a specimen of the HVP strain, 7 days after ingesting a blood meal containing 5-log PFU of WEEV per 0.1 ml blood. Notice the extensive cytoplasmic vacuolation of acinar cells, and the absence of staining

indicative of viral antigen. **(B)** Comparable section of a normal salivary gland from an uninfected control.

**Table 1 | Overall frequencies of the different types of pathology found in the midgut of four different strains of C. tarsalis infected withWEEV**.

COAV, Coachella Valley; CRS, cell rounding and sloughing; HVP, high viremia producer; KNWR, Kern National Wildlife Refuge; WR, WEEV resistant.

#### **VACUOLATION**

Arbovirus replication has been associated with the intense proliferation of intracellular vacuoles 0.3–2µm in diameter, which are thought to be major sites of viral nucleic acid replication and virion assembly (24–26). In our study, specimens were recorded as presenting vacuolation when abundant vacuoles of the appropriate size were observed in the cytoplasm of midgut cells (**Figure 1**). This type of pathology was observed in all strains in the 3-log group,and in all but the WR in the 5-log group (**Figure 6**). In agreement with previous studies (24), no vacuolation was observed in uninfected controls.

Although vacuolation was observed as early as one DPI (COAV, 5-log group; **Figure 6**), there was no particular trend in the frequency of specimens showing vacuolation over time. Furthermore, no significant differences (*P* > 0.05) were found between dose groups in the overall frequency of individuals presenting vacuolation in any strain. There were, however, significant differences between strains (*X 2* = 21.23; df = 3; *P* < 0.001), with the HVP presenting vacuolation at significantly higher frequencies than both the WR (*X* 2 = 16.19; df = 1; *P* < 0.001) and the KNWR (*X* 2 = 9.63; df = 1; *P* = 0.002).

## **NECROSIS**

In close agreement with reports by Weaver et al. (6), midgut tissue necrosis was evident as a gross degeneration of cellular integrity, to the point where midgut tissue was reduced to a thin layer consisting almost exclusively of basal lamina and traces of plasma membrane (**Figure 2**).

In experimentally infected specimens, necrosis was found only in the HVP (5-log group) and COAV (both dose groups) strains, consistently at two DPI (**Figure 6**). In these strains, no significant differences (*P* > 0.05) in the frequency of specimens showing necrosis were found between dose groups. Additionally, the overall frequency of specimens showing necrosis was not significantly different between the HVP and COAV strains (*P* > 0.05).

No signs of necrosis were observed in uninfected controls of any strain.

## **CELL ROUNDING AND SLOUGHING**

An individual was recorded as presenting CRS when midgut epithelial cells were observed to be either completely detached from the midgut epithelium or clearly protruding into the midgut lumen (6, 27) (**Figure 3**).

Among infected mosquitoes, CRS was observed in all strains and dose groups (**Figure 6**). Statistical analysis failed to reveal significant differences (*P* > 0.05) in the overall frequency of CRS both between dose groups within each strain, or between strains. Interestingly, CRS was also observed in uninfected controls of the WR and COAV strains (**Figure 6**). No significant differences (*P* > 0.05) were found between the WR and COAV strains relative to the frequency of individuals presenting CRS in infected or uninfected control groups.

CRS was not seen in uninfected controls in the HVP and KNWR strains.

#### **EGG PATHOLOGY**

Eggs were interpreted as displaying pathology when all of the following characteristics were seen together: (a) positive immunocytochemical staining, indicative of the presence of viral antigen, (b) an unusually 'smooth' yolk texture in contrast with the granular texture of the yolk in normal eggs, and (c) distortion of the chorion (**Figure 4**).

Among infected mosquitoes, the HVP strain showed signs of egg pathology in both the 3- and 5-log groups. The COAV and KNWR strains showed signs of egg pathology only in the 5 log group. No evidence of egg pathology was observed in the WR strain, or in uninfected controls of any strain (**Figure 7** and **Table 2**).

From a total of six females presenting egg pathology among all infected mosquitoes, five (83%) had been incubated for >14 DPI and one specimen (17%) had been incubated for two DPI (**Figure 7**). Statistical analysis revealed no significant differences (*P* > 0.05) between dose groups or strains in the frequency of infected individuals showing egg pathology.

## **DISCUSSION**

Pathological changes were observed in the midgut, salivary glands, and eggs of WEEV-infected mosquitoes. These changes, which included CRS, vacuolation, necrosis, and egg yolk smoothing, were

consistent with other reports of pathological effects of arboviruses in mosquito vectors (4, 6–9, 28) and contribute to a growing body of evidence that challenges the traditional belief that the impact of arboviral infection on mosquito cells is negligible (1–3).

The fact that CRS was observed in uninfected controls as well as infected mosquitoes is consistent with the notion proposed by Okuda et al. (27) that mosquitoes can regularly replace midgut epithelial cells following a blood meal, thus eliminating cells damaged by the toxic by-products of blood digestion. Others have suggested that viral infection triggers a high frequency of apoptosis and CRS in midgut cells, probably as a mechanism that modulates the viral load in this important tissue (6, 10, 29). Interestingly, uninfected controls of the WR strain presented the second highest frequency of CRS in our study (**Table 1**). Such high intrinsic turnover rate of midgut epithelial cells may enable the WR strain to eliminate and replace infected cells before they become significant foci of viral multiplication. This mechanism could, at least in part, account for the WR strain's refractoriness to WEEV infection.

Alternatively, CRS observed in specimens that did not receive an infective blood meal could be due to infection of our experimental strains with mosquito-specific viruses, which have been reportedly found in wild populations of *C. tarsalis* from various geographic locations (30).

Vacuolization and necrosis were observed only among individuals that received an infectious blood meal, suggesting that these types of pathological changes are closely associated with viral infection. Vacuolation has been reported as a result of

**Table 2 | Overall frequencies of individuals presenting egg pathology associated withWEEV infection**.

COAV, Coachella Valley; HVP, high viremia producer; KNWR, Kern National Wildlife Refuge; WR, WEEV resistant.

arthropod-borne virus replication in infected cells (24, 25), which is consistent with our observation that the highest vacuolation rates were found in the HVP strain, and the lowest in the WR strain (**Table 1** and **Figure 6**). Furthermore, the significantly lower vacuolation rates observed in the KNWR strain compared to the HVP strain (**Table 1**) suggest a lower intensity of viral replication in the former strain, and are therefore consistent with reports that found the KNWR strain to be relatively refractory to WEEV infection (13, 14). It is interesting to note that vacuolation and necrosis were in some instances observed in tissues showing no evidence of WEEV antigen presence, as indicated by the absence of immunostaining (**Figure 5**). This suggests that either tissues, which had been initially infected eventually managed to clear the virus (but the pathological effect persisted), or that these tissues are infected at levels below the detection threshold of the immuno-staining methods used. Alternatively, as was the case with CRS, we cannot rule out the possibility that these pathological changes are related to the unintentional infection of our experimental strains with mosquito-specific viruses (30).

Earlier studies by Kramer et al. (31) indicated that resistance to WEEV infection in *C. tarsalis* mosquitoes of the WR strain was intimately linked to a mesenteronal barrier, because direct injection of virus into the hemocoel resulted in infection rates and viral titers comparable to those observed in highly susceptible strains. Furthermore, it has been proposed that ultrastructural alterations of the midgut, such as those caused by the ingestion of a blood meal, can be associated with increased susceptibility to viral infection (15, 32). Therefore, it is plausible that a disruption of the mesenteronal barrier caused by an infectious blood meal (for example, the midgut tissue necrosis observed in our study) would increase the odds of virus dissemination into the hemocoel, and subsequent infection of the salivary glands. Consistent with this idea, the HVP and COAV strains (which do display midgut necrosis following an infectious blood meal) have been observed

to reach higher dissemination and salivary gland infection rates than the WR and the KNWR strains (14). In close agreement with observations by Weaver et al. (6), evidence of midgut necrosis disappeared by 72 h post-infectious blood meal, suggesting that this tissue has the ability to quickly recover from widespread virus-induced pathological changes.

Infected eggs displaying pathological changes were observed in three of the four strains used in this study (HVP, COAV, and KNWR) suggesting that WEEV infection in *C. tarsalis* eggs is not uncommon, even in unselected geographic strains of relatively recent colonization such as the COAV and KNWR. Interestingly, five out of six (83%) females presenting egg pathology had been incubated for at least 14 DPI, forcing them to retain eggs in their bodies for much longer than they would under natural conditions (<5 days post-blood meal). Although we cannot rule out the possibility that the egg pathology observed in these females is a response to the aforementioned forced retention of eggs, the positive immuno-staining observed in all eggs recorded as "displaying signs of pathology" indicates that they were indeed infected with WEEV. Therefore, it seems plausible that the pathological changes observed in these eggs are related to WEEV infection.

Additionally, it is worth mentioning that the only strain presenting egg infection in the low dosage (3-log) group was the HVP strain, which is characterized by developing unusually high WEEV titers (16). In contrast, the only strain that showed no egg infection was WR, which is characterized by its ability to maintain low WEEV titers (16). These observations suggest that a viral titer threshold must be reached in the infected mosquito before egg infection – and associated pathology – can occur.

Evidence of *C. tarsalis* egg infection with WEEV has been reported (33); however, subsequent field and laboratory studies have failed to produce evidence of transovarial transmission of WEEV in *C. tarsalis* (18, 34). Furthermore, although vertical transmission of WEEV in field-collected *Aedes dorsalis* has been reported once in the past (35), extensive efforts have failed to replicate this phenomenon in the laboratory (18, 36). Taken together, these data suggest that vertical transmission of WEEV in mosquitoes is a rather rare event.

In our study, all eggs found to be positive for WEEV antigen presented clear signs of pathological changes in their yolk and chorion, which probably rendered these eggs non-viable. Therefore, our data support the notion that *C. tarsalis* does not normally transmit WEEV transovarially (18, 34). Nevertheless, these infected eggs were surrounded by apparently healthy, viable eggs (**Figure 4**), suggesting that they could be oviposited as part of otherwise normal egg rafts. This sporadic occurrence of non-viable, virusladen eggs may explain why Thomas (33) was able to isolate virus from *C. tarsalis* egg rafts deposited by orally infected females, but could not conclusively demonstrate transovarial transmission of WEEV. Interestingly, a similar phenomenon has been observed in *C. tarsalis* infected with West Nile virus (WNV); although egg infection is frequent, trans-generational transmission occurs only rarely (W.K. Reisen, personal communication).

Romoser et al. (28), referring to the infection of *A. mcintoshi* eggs with Rift Valley fever virus, hypothesized that the oviposition of virus-laden eggs might have important epidemiological consequences, as it represents a mechanism by which viral particles could be deposited directly in the aquatic environment inhabited by mosquito larvae, which could eventually ingest these virions. The ingestion of infective viral particles during larval stages has been reported to result in transstadially transmitted infections, producing adult mosquitoes that are able to transmit virus to new hosts when they blood feed (37). Our observations of WEEV infection in *C. tarsalis* eggs are consistent with the hypothesis proposed by Romoser and his collaborators, and suggest that this process could take place in at least some mosquito/virus systems, therefore potentially playing a role in the environmental persistence of vector-borne viruses.

As noted in the results, difficulties in salivary gland tissue preparation and immuno-staining precluded a systematic assessment of salivary gland pathology at this time. However, several cases of atypical cellular morphology (vacuolation) were observed in the salivary glands of infected individuals (**Figure 5**). Salivary gland pathology associated with arbovirus infection has been found in other studies (7–9, 28, 38). Girard et al. (8, 9) have suggested that WNV-induced damage to either salivary glands or ganglia controlling salivation in *Culex pipiens and Culex quinquefasciatus* might result in reduced volumes of saliva being expectorated, which would in turn cause a reduction in feeding efficiency and the viral load injected into new hosts. Furthermore, it has been reported that long-term arboviral infections result in progressive declines in transmission rates and/or the volume of virus expectorated by infected mosquitoes (9, 13, 19). Although the instances of salivary gland pathology we observed in this study are consistent with the idea of the progressive decline of transmission rates due to damage to salivary cells, more research is needed to establish the exact role of salivary gland pathology in vector competence.

## **AUTHOR CONTRIBUTIONS**

Marco V. Neira, Farida Mahmood, William K. Reisen, Calvin B. L. James, and William S. Romoser designed the experiments. Farida Mahmood reared mosquitoes, performed experimental infections and fixed specimens. Marco V. Neira and William S. Romoser performed immunocytochemical staining, microscopical analysis, data analysis, and wrote the manuscript. All authors reviewed, edited and approved the manuscript.

## **ACKNOWLEDGMENTS**

The authors would like to thank Robert Chiles for generously providing the antibody used in this study. Emily Butler, Amanda Yant, Ehryn Rose, Carlie Rose, Regan Welch, and Kelley Vogel helped in preparing tissue samples for microscopic observation. The authors are also thankful to Joy Matthews-Lopez, Vladimir Vinogradov, and Donald Miles for their helpful advice regarding statistical analysis. María de los Ángeles López proof-read the manuscript and provided helpful comments. This project was funded in part by Research Grant 1-R01-AI39483 from the National Institutes of Health to William K. Reisen.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 July 2014; accepted: 25 September 2014; published online: 09 October 2014.*

*Citation: Neira MV, Mahmood F, Reisen WK, James CBL and Romoser WS (2014) Comparative study of the pathological effects of western equine encephalomyelitis virus in four strains of Culex tarsalis Coquillett (Diptera: Culicidae). Front. Public Health 2:184. doi: 10.3389/fpubh.2014.00184*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Neira, Mahmood, Reisen, James and Romoser. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The importance of veterinary policy in preventing the emergence and re-emergence of zoonotic disease: examining the case of human African trypanosomiasis in Uganda

## **Anna L. Okello\* and Susan C.Welburn**

Division of Pathway Medicine, Centre for Infectious Diseases, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, The University of Edinburgh, Edinburgh, UK

#### **Edited by:**

A. Paulo Gouveia Almeida, Universidade Nova de Lisboa, Portugal

### **Reviewed by:**

Lin Wang, The University of Hong Kong, China Tony Kuo, Los Angeles County Department of Public Health, USA Rafael S. Carel, The University of Haifa School of Public Health, Israel

#### **\*Correspondence:**

Anna L. Okello, Division of Pathway Medicine, Centre for Infectious Diseases, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, The University of Edinburgh, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK e-mail<EMAIL_ADDRESS>
Rapid changes in human behavior, resource utilization, and other extrinsic environmental factors continue to threaten the current distribution of several endemic and historically neglected zoonoses in many developing regions worldwide.There are numerous examples of zoonotic diseases which have circulated within relatively localized geographical areas for some time, before emerging into new regions as a result of changing human, environmental, or behavioral dynamics. While the world's focus is currently on the Ebola virus gaining momentum in western Africa, another pertinent example of this phenomenon is zoonotic human African trypanosomiasis (HAT), endemic to south and eastern Africa, and spread via infected cattle. In recent years, the ongoing northwards spread of this disease in the country has posed a serious public health threat to the human population of Uganda, increasing the pressure on both individual families and government services to control the disease. Moreover, the emergence of HAT into new areas of Uganda in recent years exemplifies the important role of veterinary policy in mitigating the severe human health and economic impacts of zoonotic disease. The systemic challenges surrounding the development and enforcement of veterinary policy described here are similar across sub-Saharan Africa, highlighting the necessity to consider and support zoonotic disease control in broader human and animal health systems strengthening and associated development programs on the continent.

**Keywords: veterinary services, neglected zoonotic diseases, public health, humanAfrican trypanosomiasis, Uganda, veterinary policy**

## **INTRODUCTION**

Human African trypanosomiasis (HAT or sleeping sickness) is classified by the World Health Organization (WHO) as a neglected zoonotic disease (NZD). Endemic across sub-Saharan Africa, HAT is transmitted to human beings through bites from the *Glossina* species of tsetse fly. Although not a novel or recently emerging disease, zoonotic HAT has been described as the "avian flu of its time" (1), associated with devastating epidemics and emergence into previously naive areas, as recently observed in Uganda (2). HAT is therefore an interesting case study through which to examine the impact of veterinary policy in mitigating the emergence and re-emergence of zoonoses into new regions; a topical issue given the unfolding Ebola crisis in parts of western Africa and other "worrying developments (*that*) show it might be time to reassess the old ideas about the distribution of these (*neglected tropical*) diseases" (3). The first purpose of this paper is to provide a short review of the history of HAT in Uganda, in order to contextualize the subsequent exploration of the factors that impede or facilitate the refinement and enforcement of veterinary policies in the country. Finally, the discussion of available options and recommendations to prevent further emergence and re-emergence

of HAT in the country can be applied to zoonotic diseases more generally across the continent.

There are two forms of HAT, their geographic boundaries roughly separated by the Rift Valley. The acute, zoonotic *Trypanosoma brucei rhodesiense* form is found in eastern and southern Africa, and the chronic, non-zoonotic *Trypanosoma brucei gambiense* form occurs throughout western Africa. Animal African trypanosomiasis (AAT or "nagana") is the corresponding syndrome in livestock, caused by various species of trypanosome also transmitted by the *Glossina* tsetse fly. Tsetse and trypanosomiasis control and eradication programs in Africa have a long history, dating from colonial times when European powers were concerned with human epidemics and the loss of animal productivity associated with the disease (4). Control efforts were largely successful up until the 1960s; however, human cases have been rising steadily since independence, with 50–60 million people across the continent currently exposed to the bite of a tsetse fly (5). Despite "political will at the highest levels" to resurrect collective action for the control of human and AAT in Africa, many feel that there is still a long way to go before the disease regains the attention it deserves and reduces to the pre-1960s level (6, 7). The intersectoral

approach required for the control of tsetse and trypanosomiasis "lies at the heart of African rural development"; shown to benefit the livelihoods of the rural poor both directly through improved health and increased nutritional outputs of livestock and indirectly through improved agricultural productivity and subsequent food security via provision of livestock outputs such as draught power and manure (5, 8).

Sharing symptoms with malaria and HIV/AIDs, HAT is often misdiagnosed or underreported by health authorities, with estimations that of the 300,000 new cases of HAT every year, only 30,000–40,000 are recorded because of issues with accessibility and quality of health services, particularly in rural areas (5). With vague first stage symptoms including recurring fever, joint pain, and nausea, the diagnosis of second stage disease is even more difficult in the absence of trained professionals, given the requirement for the trypanosome parasite to be detected in a sample of cerebrospinal fluid. Once diagnosis is made, the treatment (particularly for *T. br. rhodesiense*) is severe, with death, resulting in around 10% of cases. Without treatment, however, patients infected with both the acute and chronic forms of HAT will likely die. The current gaps in funding and technology for HAT diagnosis and treatment are a telling indicator of its "neglected" status; in the case of *T. br. rhodesiense*, no new drugs have been developed for over 60 years.

## **"A SENSE OF URGENCY": RECENT EMERGENCE OF ACUTE HUMAN AFRICAN TRYPANOSOMIASIS IN CENTRAL AND NORTHERN UGANDA**

Uganda is presently the only country to harbor foci of both the acute and chronic forms of HAT, with a focus of *T. br. gambiense* in the West Nile region to the northwest, and *T. br. rhodesiense* endemic across the southeast region of Busoga. The initial emergence of *T. br. rhodesiense* HAT in Uganda was thought to have occurred as a result of European invasion along the Congo River to Lake Victoria, with *T. br. gambiense* entering the country as a result of human migration from central to western Africa (9). Although tsetse flies have been in Uganda for "thousands of years," the public health impact and subsequent academic interest in the disease started during the early twentieth century, when it was estimated a third of Uganda's population died of acute HAT in burgeoning epidemics (9).

Progression in molecular technologies toward the end of the twentieth century established the domestic cattle population as the most significant animal reservoir in Uganda, essential for the maintenance of *T. br. rhodesiense* within human populations (10–12). Major HAT epidemics in Uganda's history have been associated with large cattle losses; over a million Ugandan cattle died in the 1890s, Rinderpest outbreaks across the country, resulting in large tracts of overgrown grazing land conducive to escalating tsetse infestations. It is thought that the tsetse flies "ran short" of cattle to feed on during this time, reverting to human blood meals, which perpetuated the spread of disease during this period (9). More recently, northwards spread of HAT has been associated with conflict and lord's resistance army (LRA) insurgence from the 1980s; people fled their homes, taking their livestock with them, which led to overgrowth of tsetse territory; "By 1990, there were only roughly one thousand cattle left in Soroti district as most of the cattle had died because of diseases, been rustled or eaten by the

armed groups in the different conflicts. . . growth of forests as a result of the war attracted the tsetse flies. . .sleeping sickness was rampant" (9).

Recent net migration of human beings and livestock back into previously overgrown areas has been attributed to this northwards spread of disease during the latter part of the 1990s (11, 13, 14). Mass rural development programs, funded by both the Ugandan government and foreign donors, were implemented upon the return of civic stability to several districts in central and northern Uganda during the 1990s. A major re-stocking exercise commenced, promoting the return of cattle and other livestock back into the area to assist the resumption of agro-pastoral activities. By 2005, however, there was a public health crisis in Uganda; molecular technologies indicated acute *T. br. rhodesiense* HAT had spread into eight new districts in as many years, with only 150 km separating the acute and chronic foci of human disease (2). The spread of disease is thought to be largely attributed to cattle movement from infected *T. br. rhodesiense* areas in the southeast of the country to previously free areas further north. It is feared that overlap of the two disease foci will spark a public health nightmare; given the parasites are morphologically similar on blood and cerebrospinal fluid smears, the only way to determine the appropriate therapeutic treatment is to know which geographical area the human patient comes from. Furthermore, the clinical effects of mixed infection, and the optimal treatment protocol, are currently unknown.

## **METHODOLOGY**

Combination qualitative research consisting of semi-structured interviews (SSIs), focus group discussions (FGDs), and observation techniques was recently undertaken in Uganda, in order to understand current veterinary policies and their level of enforcement, particularly regarding pre-movement treatment of livestock in HAT endemic areas. Key informant interviews (*n* = 13) were conducted with ministerial representatives and policy officers in the Ministries of Health and Agriculture at both the central and district government levels. In addition, field visits were undertaken in Soroti and Serere Districts in north-central Uganda, where HAT cases had recently emerged. Observations at local livestock markets complemented FGDs (*n* = 16) with farmers and local animal health officers. All key informant interviews were conducted in English, with the FGDs conducted in a mixture of Ateso and English, facilitated by a translator. The resulting transcripts were entered into Microsoft Word 2010 and manually coded according to themes, ideas, and opinions in order to develop the ensuing narratives presented in this manuscript.

## **RESULTS**

A major objective of this research was to identify and analyze the current livestock movement and disease control policies that exist in Uganda, and subsequently their level of enforcement through direct observations and data collection at the district level. The major policy concerning zoonotic disease control within animal reservoirs in Uganda is the Animal Disease Act (1918), which describes the requirements for addressing outbreaks of notifiable zoonoses such as rabies, anthrax, and trypanosomiasis (15). A separate Veterinary Public Health Act exists to promote

meat inspection and milk hygiene for the control of food-borne zoonoses such as brucellosis and bovine tuberculosis. Both acts are in need of updating, however, at present "*these are the policies that are followed as no new ones have been written post-independence*" (Informant interview, government sector).

Specifically concerning livestock movement, the official policy within the 1918 Animal Disease Act states animals cannot be moved into new areas "without clearance from veterinary officers" (15). These livestock movement restrictions were further revised after the research linking cattle movement and spread of human disease was published, resulting in new policy requiring pre-movement trypanocidal treatment of all cattle in endemic regions (16). The current procedure that should therefore be followed when cattle move from HAT endemic areas in the country's southeast is contained within Section 18 of Uganda's Animal Disease Act entitled *Rules for Infected Areas*, with particular reference to the following items:

Item 1: no stock or carcass shall be moved in or from any such area without the written permission of the commissioner of livestock and entomology or the veterinary officer or inspecting officer in charge of the area.

Item 6: no person shall leave any such area without having complied with such precautions for preventing the spread of disease as may be required by the veterinary officer or inspecting officer in charge of the area.

A second objective of the ongoing HAT research in Uganda is to establish the extent to which this existing pre-movement treatment policy is currently enforced at district livestock markets in Uganda, particularly given its importance in decreasing disease into new areas and across international borders. When asked whether they have witnessed government veterinarians treating animals at the market places, the majority of farmers indicated this was done "*from time to time*," however, some were suspicious of veterinarians under-dosing"*as the dose is very small*"(Focus Group Participants, Serere and Soroti Districts, Uganda). Most respondents signified they had to pay for treatment (between 1000 and 5000 USh) and received certificates; "*there is a doctor there, after you buy the cow, they tell us that they are treating for a certain disease, and it is a must; we cannot go without them treating our cow, it is under order*" (Focus Group Participant, Serere District, Uganda). When probed,few individuals knew what treatment their animals were receiving, justifying their lack of knowledge as "*the government vets are trying to reduce the diseases; they themselves know what they are treating for*" (Focus Group Participant, Soroti District, Uganda). Although this may be true to some extent, the current lack of community sensitization to HAT severity and its transmission is an issue; if farmers are not aware of the linkages between their cattle and *T. br. rhodesiense*, they will not see the importance for continuous post-treatment spraying, and could be further at risk of contracting the disease from their cattle in endemic areas.

A further interesting observation was made regarding the strict enforcement of livestock movement restrictions during an outbreak of Foot and Mouth Disease (FMD) in the area at the time of research (Anna L. Okello, field observations). In this way, it seems that despite the human and financial resource issues in the wider veterinary systems in Uganda, enforcement of veterinary policy is indeed possible where political motivation exists. Despite the reasons for quarantine being largely understood and respected by farmers, it was evidently placing a strain on livelihoods in the area; focus group accounts of meager cash flows as a result of livestock market closures were echoed by a private veterinary service provider in the district; "*(the quarantine) has been devastating for families and this time it has collided with going back to school – parents have no money for school fees or books, and this has been reflected in the low school attendance*" (Key informant interview, Serere District, Uganda). Some farmers explained how the lack of available animals for dowry effectively ceased marriages in the district; "*nobody is getting married – we have been told to hold off the marriage until the quarantine lifts*" (Focus Group Participant, Soroti District, Uganda).

## **DISCUSSION**

A major driver of the recent northwards movement of *T. br. rhodesiense* is the ongoing northern migration of cattle from endemic regions in the southeast, particularly since the opening up of the central and northern regions after decades of conflict (11, 13, 14). While this case study focuses on HAT, it must be remembered that this is not the only potentially fatal zoonotic disease to emerge or re-emerge as a result of infected animals entering regions where it has been previously controlled; brucellosis, rabies, porcine cysticercosis, and bovine tuberculosis are all spread through poor enforcement of veterinary policies regarding abattoir inspection, inadequate attention to disease control, and unregulated regional trade. It is for this reason that understanding the current levels of enforcement of veterinary policy, and the bottlenecks regarding this, is important.

In Uganda, a commonly cited reason for the delayed revision of the outdated veterinary policy concerns the lack of evidence – particularly prevalence data – for disease prioritization; "*you need to provide information on what the problem is, the nature of transmission, its economic and public health importance – then you can bring the stakeholders on board for their views*" (Key informant interview). For the majority of neglected tropical diseases, however, there is often difficulty in securing funds for prevalence studies in the first place; "*as much as you don't want a political crisis, we need the data for justification of spending* . . .*We are all fighting for meagre resources*" (Key informant interview).

Second, limited financial and political focus on veterinary services across much of sub-Saharan Africa has resulted in severe systemic sectoral weaknesses. For this reason, while the policy may exist, the enforcement does not, as was observed in Uganda; "*we have a very broad Animal Disease Act, which is the major policy document that directs disease control in the country, and it has provisions for most of those things. The current problem is with the implementation*" (Informant interview, government sector). Poor adherence to national policy advising pre-movement treatment of cattle with trypanocidal drugs prior to their removal from *rhodesiense*-endemic areas in the southeast of Uganda has therefore likely played a role in the northwards spread of disease in recent years. Some have also indicated the insufficient technical input

into the re-stocking movement, and its highly politicized nature – "*the re-stocking deal was done in the office of the Prime Minister*" (Informant interview, government sector) – may have inadvertently exacerbated the issue even further. This lack of opportunity for technocrats to input into the policy process has been well documented in sub-Saharan Africa (17).

At the ground level, there was further evidence that enforcement of current veterinary policy differed largely according to geographical location, likely reflecting individual commitment and available veterinary resources in that particular district. For example, while Kaberamaido and Serere Districts appeared to enforce stricter protocols surrounding pre-treatment movement of cattle, others did not; "*treatment occurred long ago but not now, we've not seen anything the past few years*" (Focus group participant, Soroti District, Uganda). The requirement for livestock movement policy enforcement is even more urgent given the propensity for farmers to prioritize cattle sales when they know international buyers and donor re-stocking programs are in operation; "*when the traders arrive from Sudan, (we) get good prices. They're taken by truck to Sudan, far away. And also the NGOs and government (for re-stocking); they give good prices, the community knows when to sell through word of mouth*" (Focus Group, Serere District, Uganda). In general, the documented field observations and farmer experiences support recently published concerns that "(Ugandan veterinary policy) implementation continues to be very patchy, with some traders and even some NGOs wholly bypassing controls" (18).

The other major observations concern the limited community level advocacy regarding the dangers of *T. br. rhodesiense* infected cattle, and the need for enabling policies that promote the required preventative animal husbandry at the farm level. In human beings, HAT's similar clinical presentation to higher profile diseases such as malaria and HIV contributes to poor public awareness; "*people are dying from sleeping sickness, but they were thinking it was HIV/AIDS, because that thing can also make you become very thin, so some people were just left to die like that* " (Informant interview Serere District, Uganda 2011). Similarly for livestock, while the various animal-specific *Trypanosome* species result in a clinically ill animal,*T. br rhodesiense* does not have any clinical impact, thus, resulting in even less desire for farmers to spend money treating what they essentially view as a "healthy" animal. The current evidence indicates that farmers prioritize tick control, given the visibility of these parasites on stock and the known impact of tick-borne diseases such as East Coast Fever (ECF). While dualpreparations that act on both ticks and tsetse flies are registered for livestock in Uganda, they are more expensive and less readily available, so smallholder farmers are less inclined to use them. Most farmers interviewed purchase the cheaper tick-only preparations that have no impact on*Trypanosome* transmission, consistent with previous findings by Bardosh et al. (19). Improving the supply and economic justification from both the human and animal health perspective for why farmers should use tsetse-tick preparations would be an important way forwards for the Ugandan veterinary sector, and one that is recommended by the authors.

Finally, it is interesting to note that the Animal Disease Act can be enforced for trade diseases such as FMD, despite in this case the socioeconomic impact of quarantine appearing arguably greater than the disease itself, given the limited access of smallholder farmers to formal livestock markets. Considering "pro-eradication" policies for the control of trade diseases such as FMD are able to remain strong in this way, it is vital that the same attention is turned to zoonoses control in endemic countries, to prevent further emergence into naive areas or across international borders.

Through documenting the recent observations of HAT control in Uganda, we have highlighted the role of the veterinary sector in zoonoses prevention, presenting a case for greater economic and institutional focus on the African veterinary sector as a whole. However, long distances, poor infrastructure, and limited cash flow/disposable income all impact the provision and utilization of quality human and animal health services across much of sub-Saharan Africa. This ultimately affects disease reporting, impacting the level of attention placed on a disease issue by policy makers (20). Furthermore, even where policy actors do accept the need for zoonoses control, the issues of policy development and enforcement are compounded where multiple sectors are involved, further impeding the timely adoption and implementation of innovative policy processes for zoonoses control (17). It is for this reason that veterinary sector improvements need to consider the various external economic and developmental bottlenecks to policy development, enforcement, and control that could impact disease control more generally on the continent.

## **CONCLUSION**

There are several ongoing policy issues regarding livestock movement and disease control in Uganda, stemming from a variety of social, economic, and political factors. Up to date, enforceable veterinary policy has the potential to significantly contribute to the economic development of a country via joint impacts on human and animal disease control, improved production outputs that impact regional markets, and access of the human population to improved nutrition. Despite this,the importance of veterinary policy is often underestimated by the health and development sectors alike, particularly regarding its role in the prevention of emerging and re-emerging zoonotic disease, as described by this case study.

Weak enforcement of a veterinary policy directive that simultaneously controls a fatal human disease and improves livestock productivity questions the type of evidence, advocacy, and capacity required to ensure zoonoses control efforts in animal reservoirs are realized. We need to overcome this disabling triad of capacity, advocacy, and evidence in order to move beyond our relatively limited understanding of zoonoses transmission – and its implications in changing agricultural contexts – toward a fuller, holistic, and more responsive veterinary system of analysis, intervention, and control. Awareness of the potentially harmful human diseases carried by domestic animals and wildlife is vitally important, not only just for the communities that face daily exposure to them but also for the policy actors and key decision-makers that play a role in preventing the emergence and spread of zoonoses at the crossroads of human health, agriculture, and development.

## **REFERENCES**

1. Okello AL,Bardosh K,Smith J,Welburn SC. One health: past successes and future challenges in three African contexts. *PLoS Negl Trop Dis* (2014) **8**(5):e2884. doi:10.1371/journal.pntd.0002884

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 July 2014; accepted: 16 October 2014; published online: 03 November 2014.*

*Citation: Okello AL and Welburn SC (2014) The importance of veterinary policy in preventing the emergence and re-emergence of zoonotic disease: examining the case of human African trypanosomiasis in Uganda. Front. Public Health 2:218. doi: 10.3389/fpubh.2014.00218*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Okello and Welburn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

REVIEW ARTICLE published: 03 December 2014 doi: 10.3389/fpubh.2014.00265

## **Servio Urdaneta-Morales\***

Laboratory for the Biology of Vectors and Parasites, Tropical Zoology and Ecology Institute, Central University of Venezuela, Caracas, Venezuela

#### **Edited by:**

A. Paulo Gouveia Almeida, Universidade nova de Lisboa, Portugal

#### **Reviewed by:**

Ana Gonçalves Domingos, Universidade nova de Lisboa, Portugal Maria Odete Afonso, Universidade nova de Lisboa, Portugal

#### **\*Correspondence:**

Servio Urdaneta-Morales, Laboratory for the Biology of Vectors and Parasites, Tropical Zoology and Ecology Institute, Central University of Venezuela, Los Próceres Street, Caracas 0058 0212 9917550, Venezuela e-mail<EMAIL_ADDRESS>
The unprecedented emergence of important public health and veterinary zoonoses is usually a result of exponential population growth and globalization of human activities. I characterized Chagas' disease as an emergent zoonosis in the Caracas Valley (Venezuela) due to the following findings: the presence of reservoirs (Didelphis marsupialis, Rattus rattus) and vectors (Panstrongylus geniculatus, Panstrongylus rufotuberculatus) infected with Trypanosoma cruzi in urbanized or marginalized areas; the elevated contact between P. geniculatus and human beings detected by parasitological and molecular examinations of triatomine feces demonstrated the possibility of transmission risks; a study of outbreaks of urban Chagas' disease reported the first proven case of oral transmission of T. cruzi to human beings; the risk of transmission of glandular metacyclic stages from marsupials by experimental ocular and oral instillation; mice genitalia infected with T. cruzi contaminated blood resulted in the formation of amastigotes very close to the lumen suggesting that there may be a possibility of infection via their release into the urine and thence to the exterior; the ubiquitous histotropism and histopathology of T. cruzi was demonstrated using a mouse model; the presence of experimentalT. cruzi pseudocysts in adipose, bonecartilage, and eye tissue indicated a potential risk for transplants. Socio-sanitary programs that include improvements in housing, vector control, and access to medical treatment, as well as strategies aimed at combating social inequalities, poverty, and underdevelopment should be undertaken in those areas where zoonoses are most prevalent. Disciplines, such as Ecology, Epidemiology, Medical Entomology, Human and Veterinary Medicine, Environmental Studies, Public Health, Social and Political Studies, Immunology, Microbiology, and Pharmacology could all provide important contributions that aim to reduce the occurrence of factors governing the spread of emergent diseases.

**Keywords: Chagas' disease, emerging urban zoonosis, Caracas Valley (Venezuela)**

## **INTRODUCTION**

American trypanosomiasis (Chagas' disease), a metaxenic zoonosis caused by *Trypanosoma (Schizotrypanum) cruzi* Chagas*,* 1909, is endemic to Neotropical and Nearctic regions (Salt Lake City, 41° N in USA/56° S in the argentine Patagonia and northern Chile as well as the Caribbean Islands). A total of 8 million people in 21 countries are infected by the disease with mortality rates of approximately 10,000 per year. This parasitosis is found in particular landscapes inhabited by mammal reservoirs and insect vectors (Hemiptera, Reduviidae, Triatominae), which together make up the wild cycle, the most primitive zoonotic transmission dynamic, in which the parasite travels between more than 200 species of mammals from seven orders and about 140 species of insects in 15 genera (1, 2).

The most ancient reservoirs in this cycle are the Eutherian (Cingulata, Dasypodidae: armadillo); Rodentia (Echimyidae); and Metatherian (Didelphimorphia, Didelphidae: opossum) (3, 4). These synanthropic mammals have migrated from their natural ecosystems to human communities, while at the same time, human beings have encroached onto their habitats. In both cases, *T. cruzi* infects individuals giving rise to the so-called peridomestic and domestic Chagas' disease cycles. This accidental anthropozoonosis may develop in rural as well as urban areas and is conditioned by both environmental and social elements (1, 5). In Venezuela, *T. cruzi* circulates between 39 species in the Marsupiala, Edentata = Xenartra, Chiroptera, Carnivora, Rodentia, Primates, and Lagomorpha and is transmitted by 22 insect vectors. The main vector is *Rhodnius prolixus* due to its broad distribution (over nearly three quarters of the total land area of Venezuela) and its high domiciliation (6–8).

*Trypanosoma cruzi* is a hemoflagellate (Protozoa, Kinetoplastida, Trypanosomatidae) with two developmental stages: extracellular bloodstream trypomastigotes and intracellular tissue amastigotes. In vectors, both forms are extracellular: epimastigotes that develop in the lumen of the midgut, and metacyclic trypanosomes found in the rectal part of the hindgut. The latter of these two forms is the infective stage, and transmission of the parasite primarily occurs when contaminated feces evacuated by the parasitized insect come into contact with healthy mucosa or the damaged skin of the host. In contrast, in the African trypanosomes and the Neotropical *Trypanosoma rangeli,*the parasites initially develop in the gastrointestinal tract of the insect vector and from there migrate by tropism to the salivary glands where the infective stages develop before being transmitted mechanically when the insect bites a host. In both cases, the*Trypanosoma* species cause zoonotic parasitosis in human beings (9, 10).

American trypanosomiasis discovered in Brazil by Chagas (11), was demonstrated in Venezuelan human beings by Tejera (12), and Pifano (13) described the characteristics of the environmental niches of this zoonosis as well as the features of the principal bioregions where it develops.

## **DEFINITION OF EMERGING INFECTIOUS DISEASES AND ZOONOSES**

The emergence and spread of smallpox during the "discovery" of America in the 16th century, which attacked vulnerable nonimmune indigenous populations and the sudden appearance of the great pandemics: bubonic plague, cholera, typhus, syphilis, and leprosy in Europe during both the Middle Ages and the Modern period are all examples of emerging diseases. These were triggered by massive migrations from rural to more-developed areas resulting in overcrowding and social changes, which together with a lack of hygiene exposed populations to transmissible agents (14).

Among the many, and often divergent, definitions of the concept of emergent infectious diseases, two appear to be the most appropriate as they are based on the dynamics of several elements that act synergistically to produce the diseases. The first of these is "Infectious diseases are said to be emergent when qualitatively unexpected phenomena resulting from local interactions appear; this tends to occur suddenly" (14). The other is that emerging infectious diseases are infections that have newly appeared in a population, or already exist but whose incidence is rapidly expanding over a geographical, host or vector range, or have been reported in new populations (15–18). Thus, every infectious disease is emergent until it reaches endemic status. These broad definitions encompass a range of human diseases all of which represent a significant threat to public health.

Zoonoses are infectious diseases that are transmitted between their natural hosts and human beings (19).

## **PURPOSE OF THE REVIEW**

The principal determining factors that make up the web of causation of the infectious diseases can be summarized as follows: biological (infectivity and pathogenicity) and molecular properties of the pathogens; characteristics of the vertebrate hosts; vector competence and migration, anthropogenic factors causing significant damage to ecosystems resulting in the alteration or destruction of mammal hosts and insect vectors habitats as well as changes in their behavior; and decrease in the funding allocated to health authorities for their surveillance, prevention, and control. While a few infectious diseases have been eradicated (smallpox) or controlled (dracunculiasis, measles, polio), it is probable that most of the rest of them will not be, at least in the short term, because the causal factors for their emergence remain. This being so and faced with the fact that the recurrence rate will not only continue but also increase, new approaches and tools must be found and implemented in order to at least prevent the establishment

of these diseases (10, 15–20). This article was stimulated by these considerations.

The studies based in the Caracas Valley will be compared with reports of emergent diseases produced by zoonotic agents from other countries. The Caracas Valley was selected because it is still classified by the health authorities in Venezuela as "not endemic" and is, therefore, are not included in the Venezuelan National Control Programme (21).

## **CHARACTERISTICS OF THE CARACAS VALLEY**

Caracas, the capital city of Venezuela, lies in a depression within the coastal mountain range in north-central Venezuela. The city is located at 960 m.a.s.l. and comprised five municipalities and two federal entities: the Capital district (Libertador municipality) and Miranda state[] (Chacao, Baruta, El Hatillo, and Sucre municipalities). All these municipalities together make up the Caracas Metropolitan district (10° 230 1800–10° 340 0000 N; 66° 510 3000– 67° W 100 3500) (**Figure 1**). The Caracas valley has an altitude of 870–1043 m.a.s.l., average temperature 22°C, and annual rainfall of 870 mm; the natural vegetation is pre-montane forest. The population density of Caracas has been increasing exponentially due to steady migration from other parts of Venezuela, as well as neighboring countries, and is now fully urbanized. This migration has resulted in a population with minimum hygiene standard as well as poor socioeconomic conditions. Much of this population is housed in urban slums, located on the banks of the numerous rivers that cross the city as well as in the neighboring savannas and mountains forming a regrettable belt of misery. The slums adjoin built-up medium and high value residential or commercial areas and similar situations have developed in other areas along the valley, all of which have profoundly modified the natural environment. The Caracas Valley is, thus, an excellent example of the progressive modification of an ecosystem, where an increasing human population has invaded a region already populated by Chagas' disease insect vectors and mammal hosts (21, 22).

**of the Metropolitan District**.

#### **Table 1 | Histopathology in organs of white mice infected with isolates ofTrypanosoma cruzi from urban Panstrongylus geniculatus**.

X, moderate; XX, marked; XXX, intense.

## **CHRONOLOGICAL DESCRIPTION OF INVESTIGATIONS INTO THE CHAGAS' DISEASE WEB OF CAUSATION IN THE CARACAS VALLEY THAT DEMONSTRATE ITS STATUS AS AN EMERGING URBAN ZOONOSIS**

The research undertaken by Pifano (22) was the first to demonstrate the presence of Chagas' disease in the Caracas Valley. This was done by identifying the components of the transmission cycle as follows: reservoirs (*Didelphis marsupialis*) and vectors (*Panstrongylus geniculatus*) captured in slums and housing estates were found to be infected by *T. cruzi* by examining fresh blood samples and smears stained with Giemsa, hemoculture, xenodiagnoses, and histopathology of opossums, as well as the examination of vector fecal samples.

The author suggested that the omnivorous/predatory diet of these marsupials, which feed on the reservoirs and vectors of *T. cruzi*, increases the probability of their becoming infected by this heteroxenous parasite. This, together with their synanthropic nature has enabled them to easily adapt to anthropically modified environments frequently invading areas inhabited by human beings. Furthermore, vectors showed positive, by immunodiffusion, for human and animal blood.

The tissue ubiquity and the pathology caused by *T. cruzi* obtained from *D. marsupialis* were determined in this host and experimental NMRI mice (23–26) (**Table 1**).

Deane et al. (27) described a unique property of *D. marsupialis*, whereby the *T. cruzi* morphotypes typically found in the intestines

of its vectors, were also observed in the anal scent glands of this species. This property has been reported from wild *D. marsupialis* and *Didelphis albiventris* in both Brazil and Venezuela (28–30). In addition, the experimental colonization of *D. marsupialis* and *Lutreolina crassicaudata* glands was produced by s.c. injection of bug urine and fecal material (31–33).

Urdaneta-Morales et al. (34, 35) described this *T. cruzi* luminal cycle from naturally infected *D. marsupialis* captured in highly urbanized areas within the Caracas Valley. Numerous epimastigotes of different sizes, most undergoing binary division giving rise to rosettes, intermediate forms, and metacyclic trypomastigotes infective to mammals, were observed. These glands, thus, provide a favorable environment for the development of the *T. cruzi* vector cycle.

Furthermore, glandular material inoculated s.c., i.p., or instilled orally and ocularly in healthy opossums and white mice caused parasitemias with the pleomorphic forms typical of the parasites as well as intracellular parasitism with the multiplication of amastigotes in cardiac and skeletal muscle (**Figure 2**). Glandular metacyclics from LIT (liver infusion tryptose) cultures inoculated into these mammals gave the same results. Infection caused 100% mortality in the mice, whereas all the opossums survived. Nymphs of *R. prolixus* fed on both hosts showed infective metacyclics in the feces (34, 35).

Herrera and Urdaneta-Morales (36) captured 37 *Rattus rattus*, one *R. norvegicus*, and nine *Mus musculus* in urbanized areas (Colinas de Bello Monte, Los Chorros, El Cafetal, Las Acacias, San Román, Parque del Este and Caricuao) in Caracas. Of these, conventional examinations of fresh blood and smears stained with Giemsa, and xenodiagnosis revealed the presence of *T. cruzi* stages in two *R. rattus* individuals (**Figure 3**). Tissue sections from *R. rattus* showed numerous pseudocysts with amastigotes in the heart as well as moderate parasitism of the skeletal muscle and smooth muscle of the duodenum. All mice inoculated with xenodiagnosis' bug fecal material showed a moderate pattern of tissue tropism in the same organs as in well as in the colon and pancreas (**Figure 4**). *Trypanosoma* (*Herpetosma) lewisi* was also detected in infected rats, but not in the other rodents examined.

Reyes-Lugo and Rodríguez-Acosta (37) found *P. geniculatus* colonizing the interior of a well-built house in an area of transition between cloud forest and humid montane woodland in the mountainous region of Hoyo de la Puerta (Miranda state) on the outskirts of Caracas. A total of 20 *P. geniculatus* specimens in all stages of development gorged with blood were shown to be infected by *T. cruzi*. The authors also found *R. rattus* in several tunnels connecting the inside floor of the house with the outdoors, in which several *P. geniculatus* individuals and their eggs were found. The authors suggested that human activities have led to the disappearance of the natural habitats of this triatomine and with them its food sources, thus favoring its domiciliation. This situation has been described by Reyes-Lugo (38) in a further eight localities in the center-north of Venezuela, including the Caracas Valley.

Xenotransplantations have demonstrated zoonotic infections produced by viruses, bacteria, protozoa, fungi, and helminths, thus showing their importance as risk factors for these diseases (20). Using a mouse model, the possibility of the transfer of *T. cruzi*

**FIGURE 2 |Tissue sections showing pseudocysts containing amastigotes (H-E)**. **(A)** Heart of Didelphis marsupialis infected orally by glandular material cultured in LIT medium (400×; **(B)** muscle layer of anal gland of opossum, infected as above (1000×); **(C)** heart of mouse infected intraperitoneally by glandular material from a naturally infected opossum (400×).

was determined in organs often used during these procedures. Isolates from *D. marsupialis* and *R. rattus* captured in Caracas were inoculated in adipose, bone-cartilage, and eye tissue, observing the intracellular presence of the parasite in all cases (39–41) (**Figures 5**–**7**). This constitutes an alternative transmission pathway, whereby natural *T. cruzi* intracellular multiplication could be enhanced in immunosuppressed hosts. Isolates of *T. cruzi* stages found in the eye tissue of mice produced an electrophoretic band pattern that identified the parasite as TcI (ZI).

Based on several references cited by Zeledón (42) regarding the presence of *T. cruzi* trypomastigotes in opossum (*D. marsupialis)* and mouse urine, together with the suggestion of Dias (43) that the presence of these stages in the chagasic female's menstrual blood

**FIGURE 3 | Flagellate stages ofTrypanosoma cruzi**: **(A)** stout bloodstream trypomastigote from naturally infected Rattus rattus (Giemsa, 1400×); **(B)** metacyclic trypomastigote from feces of Rhodnius prolixus used for xenodiagnosis of naturally infected R. rattus (Giemsa, 1400×).

could enable transmission through coitus, Herrera and Urdaneta-Morales (44) carried out the following investigation: the blood of mice infected with *T. cruzi* isolates from *R. rattus* was instilled into the urinary meatus (females) and penis (males) of healthy mice, and a *T. cruzi* isolate from a human being was inoculated into the scrotum. The genital instillations resulted in the invasion of the heart, skeletal muscle, duodenum, pancreas, sternum, ovary, testis, and vas deferens. In addition, scrotal inoculation caused the invasion of the liver, spleen, lung, kidney, urinary bladder, and seminal vesicle (mucosa close to the lumen) (**Figure 8**).

Carrasco et al. (21) established that there is a high risk of the transmission of Chagas' disease in the Caracas Valley due to the contact between *P. geniculatus* and human beings. Natural infection by *T. cruzi* was determined by examinations of fresh and stained stools. In addition, the random amplification of polymorphic DNA for parasite identification and group typing, and a dot-ELISA test to identify the gut content of the triatomine bugs showed that 66% of the 88 triatomines studied were infected by TcI, of which 60% reacted positively to human antiserum. The relationship between the percentages of bugs with fecal contents reactive to human antiserum and those reactive to all the antisera used was 98%, while approximately 41% of the bugs fed on human beings were infected by *T. cruzi*.

The first proven cases of the transmission of *T. cruzi* to human beings occurred in urbanized areas within the Caracas Valley (Chacao municipality and Antímano parish). These outbreaks represent two of the most numerically important cases of orally acquired Chagas disease in Latin America to date. A total of 124 patients in the acute phase of infection were characterized by their clinical symptoms, electrocardiograms, immunoenzymatic tests, indirect hemagglutination, and PCR. Examinations of blood, hemoculture, and inoculation in a mouse model were also performed. Polluted guava juice was statistically shown to be the only source of infection and the person who prepared the juice was

**FIGURE 4 | Histological sections showing pseudocysts ofTrypanosoma cruzi with amastigotes in (A) cardiac tissue of naturally infected Rattus rattus (H-E; 560**×**); (B)** skeletal muscle of naturally infected R. rattus (H-E, 560×); **(C)** smooth muscle fiber from the colon of an experimentally infected mouse (H-E, 960×); **(D)** acinar cell of pancreas of experimentally infected mouse (H-E, 1400×).

infected with *T. cruzi*. The presence of infected *P. geniculatus* and *R. rattus* was confirmed in the slum where this individual lived (45, 46).

The capture of two specimens of *Panstrongylus rufotuberculatus* infected with *T. cruzi* inside houses in the town of El Hatillo, close to Caracas (originally a forested area but now completely urbanized), was reported by Zavala-Jaspe et al. (47). One of the isolates was characterized by the following: parasitological examinations of the intestinal contents, infection of a mouse model, hemoculture, xenodiagnoses, and *in vivo* and *in vitro* metacyclogenesis producing morphotypes characteristic of the parasite; infection by *T. cruzi* was confirmed by PCR.

All of these chronologically described results constitute important epidemiological risks for public health in this capital city.

## **DYNAMIC OF EMERGENT DISEASES: TRANSMISSION PATHWAYS-DISEASE TRANSMISSIONS**

Until the end of the 80s, it was thought that emergent diseases had been eradicated or that they were limited to underdeveloped areas. This was due to the fact that the biomedical control programs were abandoned, which resulted in a huge spate of emerging diseases. The situation was aggravated by the failure to isolate or identify the pathogens responsible, coupled with the difficulty of characterizing the symptoms due to their (in the majority of cases) extensive pre-patency and subclinical nature. Research into the global occurrence of important outbreaks led to the conceptualization of the

**FIGURE 5 | Amastigotes and intermediate stages (arrows) ofTrypanosoma cruzi in (A,B) perifery cytoplasm of an adipocye**; **(C)** cytoplasm of immature adipocite (preadipocite); **(D)** intercellular substance in connective adipose tissue; **(E)** parasitized macrophage located between uninfected adipocytes (H-E; 1400×).

causes of the emergence of these diseases (15, 19). In both excellent reviews of the many risk factors that influence the emergence of infectious diseases according to their pathogens, an estimated 75% of these diseases, principally viral, bacterial and parasitic, and possibly vector-borne, have emerged over the past two decades from a wildlife source.

Given that each reservoir and vector species occupies a specific ecological niche and shows a particular behavior pattern, the physical and biological environments should be maintained in a dynamic equilibrium with human populations and society in order to prevent the emergence and spread of new diseases. It is important to realize that the emergence of these diseases is most likely to occur after changes to the components of this dynamic equilibrium. Consequently, zoonoses (including the parasitic ones) and particularly emergent zoonoses have sparked interest in the international scientific community. This is due to their increasing importance to human and animal health as they cause high indices of morbidity and mortality in both rural

and urban areas and in endemic and non-endemic areas, with huge repercussions for the economies and health of the countries affected (5, 48–52).

Human beings, in their struggle for survival, have profoundly modified the natural environment, adapting it to their needs as they colonize different landscapes and habitats, all of which provide natural niches for pathogens responsible for zoonotic infections. These can derive from, and become emergent in, many different environments: wild, rural, regional-urban, and even globalurban. These niches house an enormous number of vertebrates and invertebrates that, in turn, host a huge variety of macro- and micro-pathogens; the risks of transmission to human beings are obvious (5).

Anthropogenic changes (environmental changes, deforestation, reforestation, road construction, urban growth, trade, translocation, and keeping of exotic pet, ecotourism, consumption of contaminated water or raw or undercooked food in order to retain its flavor and nutrients) are those that have proven to cause the

**FIGURE 6 | Pseudocysts with stages (arrows) ofTrypanosoma cruzi in sternum of mice experimentally infected with different isolates**. **(A)** Perichondrium with nests showing amastigotes and intermediate stages in chondroblasts (400×; HE); **(B)** same stages in stroma of perichondrium and in the cytoplasm of a macrophage (1000×; HE); **(C)** and **(D)** two broken chondrocytes with several amastigotes and one flagellated stage (400× and 1000×, respectively; HE); **(E)** several amastigotes in a osteocyte (1000×; HE); **(F)** pseudocyst with amastigotes in the bone marrow (1000×; HE).

most stress on the balance between the physical and biological environments intrinsic to the pathogen-host-vector association.

The key factor for the unprecedented levels of these emergent diseases is the synergism between two circumstances, the globalization of human activities with the exponential increase in the population growth rate causing progressive deterioration of the relationship between human and environmental health, and therefore modification and destruction of the natural niches of reservoirs, vectors, and pathogens. Unprecedented increases in the incidence of infectious emergent diseases and the extinction of species with the resulting loss of biodiversity are examples of this. Immune pressure and the abuse in antibiotic use also cause highly frequent changes in pathogens that are expressed as mutations in their virulence, pathogenicity, and genetic structure. Another important characteristic worthy of mention is the generalist nature of the pathogens that affect human beings: those that are capable of infecting multiple hosts, especially when they belong to more than one order, are highly likely to infect human beings. An excellent example of this type of pathogen is *T. cruzi* that, as already mentioned, parasitizes species of mammals from 7 orders and triatomines from 15 genera (10, 15–20, 48–61).

## **NEGLECTED ZOONOSES**

The critical situation regarding infectious diseases that persists in many indigenous populations in several continents must be highlighted. These zoonoses have a higher prevalence in regions between latitudes 35°N and 35°S, with altitudes below 2200 m and temperatures ranging from 15°C to 40°C. These populations are some of the world's poorest, most anonymous and ignored. They have been subjugated by foreign powers that "discover" them, conquer and enslave them; they have suffered unjust discrimination and political and economic exclusion. Multinational corporations have, among many other things, denied them their rights to education, their sources of cultural and physical survival, their traditional knowledges, the ownership of some of the most biologically diverse territories in the world thus producing local or global extinctions of species, and their languages, in short, their ethnic cultural identity.

Emerging diseases are considered as "neglected" because the huge investments resulting from The Millennium Declaration, adopted by the United Nations in September of the year 2000 and applied at a large scale to projects for the prevention and/or control of HIV/AIDS, malaria and tuberculosis (the "big three"), have not up until now been used for combatting them. Many scientists, institutes for health and pharmaceutical companies continue to ignore and exclude them. The argument put forward for this is that there is no reliable information about the health burdens of these "low hanging fruit" populations. This lack of knowledge has led to a reduction or elimination of funding and research within these marginalized communities. All this, in spite of the fact that millions of people from Sub-Saharan Africa, Asia, Latin America, and the Caribbean continue to suffer diseases at least as (if not more) serious as the three mentioned (62–68).

## **PROGRAMS FOR THE PREVENTION AND CONTROL OF ZOONOSE**

The great diversity of risk factors that produce the emergence, reemergence and, in many cases, persistence of zoonoses caused by pathogens that are already known, as well as those that have only recently been characterized, or have undergone changes in their bio-ecological characteristics in poor communities are particularly notable in countries governed by strong socio-politico-religious groups, as is the case for Africa, Asia, and also Latin American and Caribbean countries whose populations are indigenous or of African descent.

By other hand, most of the alterations in the behavior, cognitive or psychosocial patterns of the indigenous populations infected with zoonoses are, in general, similar even between communities from different continents, it is appropriate to mention them here. The *per se* knowledge that they are ill, or if they are told that they have a disease of this nature, produces symptoms that begin with slight depression, before developing into anxiety, stigma, and

alexithymia. Their concern that the disease will worsen, the impossibility of finding a cure and their fear that they will infect others, distances them from their family circle and friends, while the weakening of their immunological systems only serves to worsen their condition (68–70).

Currently (63–65), a comprehensive framework model is being applied in some continents with promising results. This model attempts to control some diseases and reduce poverty with a view to returning the basic rights of these populations (equality, ethical treatment, and health) stolen from them. According to specialists, a key strategy for minimizing the deficiencies in funding is to try

to increase the commitment of the family and local community so that they themselves help to prevent, control, and even eliminate zoonoses through feasible and economic methods. Some examples of such community-based approaches are Community-Led Total Sanitation (CLTS), which has completely eliminated open defecation resulting in a massive reduction in the incidence of enteric diseases; controls in China for reducing the rate of transmission of *Schistosoma japonicum* from infected bovines and human beings to snails; management of sleeping sickness in Uganda caused by *T. rhodesiense* by chemotherapy of bovine reservoirs en masse and the use of insecticides for the control of the insect vectors.

**FIGURE 8 | Histological sections from albino mice intravaginally instilled with a strain ofTrypanosoma cruzi from Rattus rattus, and scrotally inoculated with a strain from a human patient, showing nests (arrows) with amastigotes in (A) cardiac tissue;**

**(B)** skeletal muscle; **(C)** pancreas (acinus); **(D)** liver; **(E)** urinary bladder (epithelium very close to the lumen and lamina propria); **(F)** seminal vesicle (mucosa close to the lumen). [**(A,B,D)**: l400×; **(C,E,F)**: 950×; H-E].

Molyneux and Malecela (66) drew up a set of macro-priorities and recommendations with the idea of clarifying these objectives as listed below (**Table 2**). These authors emphasize the scarce knowledge that clinicians and policy makers, general speaking, have when they diagnose for example a zoonosis with no specific symptoms such as Brucellosis, Leptospirosis, Rickettsiosis, or Q fever, as Malaria, as well as their ignorance of the possibility that

zoonotic infections can, in the long term, cause cancer (Trematodiases) or Neurocysticercosis. All this as a consequence of the absence of microbiological and molecular tools in laboratories and hospitals, due to the financial limitations they suffer; further evidence of the sad neglect these populations face.

With this in mind, strategies that should be developed include primary health education in conjunction with the health sectors

## **Table 2 | Macro research priorities identified by DRG6 (Disease Reference Group – WHO UNDP World Bank Special Programme)**.

of competent indigenous organizations; the translation of books into indigenous languages, training for indigenous health workers, teachers and community leaders with an emphasis on teaching about the main risk factors, and the time of year these are most prevalent; constant entomological and serological monitoring, after training, by community leaders; the implementation of vector control methods, such as indoor residual spraying pyrethroid and improving housing; safe preparation of food for its immediate

consumption or transport; safe water for drinking and basic sanitary conditions and the monitoring and treatment of infected individuals. In addition, surveillance and the use of drugs and vaccines, research in natural products; clinical and epidemiologic research; use of electron microscopy, genomics/bioinformatics, and applied biotechnology should be available to ensure healthier lives. All these aspects should occur within the context of environmental sustainability, and all are necessary in the regions where most of the zoonoses are found. There is an urgent need for a concerted effort by disciplines such Ecology, Epidemiology, Medical Entomology, Human and Veterinary Medicine, Public Health, Environmental Studies, Immunology, Microbiology, Pharmacology, Social and Political Studies, and Anthropology (62, 69–77).

 This list of emergent–reemergent pathogens should, like any other, be periodically revised and updated given the dynamics of the factors that determine changes in its transmission pathways.

Following on from this, it is appropriate to discuss the following considerations as a consequence of the permanent rise in industrialization and urbanization in Latin America and the Caribbean Region, the transmission of several infectious diseases, has increased dramatically (4, 5, 20, 55, 64, 69, 78–83). Its diagnosis, however, can be delayed for several months depending on the technique used. This obviously delays treatment causing the patient's condition to worsen. The fact that *T. cruzi* and its vectors can remain viable in food over long periods of time only exaggerates this problem. Nevertheless, these situations can be averted with the use of newly developed molecular tools, which, together with reference datasets, have shortened the diagnosis time to only a few days (45, 46, 84). It is to be hoped that in the future, some of the risk factors of these diseases will be at least qualitatively and quantitatively reduced, if not eliminated. Several of these measures could seem utopic because of the small attention they have traditionally received, but it would be a mistake not to mention them. The current healthcare system, although insufficient, should at least be used to improve the situation of the poorest communities while other tools are being developed. The need for investigation into the use of drugs to combat the infections, while minimizing the risks of the evolution of pathogen resistance to them, is obvious (85).

The investigations undertaken in the Caracas Valley, presented and discussed using a bio-eco-epidemiological approach, show that Chagas' disease is most certainly an emerging urban zoonosis and underline the high risks of infection for human beings and their domestic animals.

The following is a summary of the most important epidemiological aspects of the public health situation regarding the transmission of Chagas' disease by *D. marsupialis* in the Caracas Valley, which taken together create conditions of permanent risk from zoonotic infection by *T. cruzi* in areas with or without the presence of vectors: the adaption of *D. marsupialis* to city environments, from slums to economically wealthy urbanized zones; the capacity this marsupial has to infect mammals with metacyclic trypomastigotes that develop in the lumen of the anal scent glands; the violent ejection of fluid from the scent glands in response to threat and the proximity of these glands to the rectum and the urogenital organs, thus providing a means by which the feces and urine can become contaminated with the glandular fluid containing the metacyclics, leading to the possibility of infection through the consumption of spoiled food and drink.

The finding that contact between *T. cruzi* bloodstream trypomastigotes and genital mucosa can produce blood and tissue infections through the formation of pseudocysts with amastigote stages close to the lumen of the urinary bladder and seminal vesicle suggests an alternative possibility for the transmission of this zoonosis; when the cysts rupture, they could liberate the parasites either into the genital secretions or urine and thus to the exterior.

The fact that *R. rattus* may also act as a reservoir for *T. cruzi* and the evolution of the adaption of *P. geniculatus* and *P. rufotuberculatus* to human dwellings increase and geographically widen the risks for the transmission of Chagas' disease in the Caracas Valley.

The infection of adipose, bone-cartilage, and eye tissues by *T. cruzi* demonstrates their role as important micro-reservoirs of this parasitic flagellate and reveals yet another transmission pathway during organ transplants in immunosuppressed patients.

This review was initially undertaken in response to the risks of transmission brought about by the close contact between *D. marsupialis*, *P. geniculatus,* and human beings along the whole of the Caracas Valley, and the report (for the first time) of 124 acute cases of proven *T. cruzi* transmission to inhabitants in a totally urbanized sector of this capital city, as well as important results from other investigations carried out in areas encompassing the poorest slums to zones of high economic value. The permanent and high levels of industrialization and the migration of people from endemic regions of Venezuela and other countries can only further increase the probability of the emergence of this zoonosis.

## **ACKNOWLEDGMENTS**

The author is most grateful to Dr. Frances Osborne, Tony Barrucci, and Javier Pinto for aid in preparing the manuscript. We would like to thank the reviewers for their suggestions and insightful comments.

## **REFERENCES**

soil-transmitted helminths among school children in Honduras. *Parasit Vectors* (2014) **7**:354–69. doi:10.1186/1756-3305-7-354

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 July 2014; accepted: 14 November 2014; published online: 03 December 2014.*

*Citation: Urdaneta-Morales S (2014) Chagas' disease: an emergent urban zoonosis. The Caracas Valley (Venezuela) as an epidemiological model. Front. Public Health 2:265. doi: 10.3389/fpubh.2014.00265*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Urdaneta-Morales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Trypanosoma cruzi, the causal agent of Chagas disease: boundaries between wild and domestic cycles in Venezuela

## **Leidi Herrera\***

Laboratory of Parasite and Vector Biology, Institute of Tropical Zoology and Ecology, Science Faculty, Central University of Venezuela, Caracas, Venezuela

#### **Edited by:**

Juan-Carlos Navarro, Central University of Venezuela, Venezuela

#### **Reviewed by:**

Omar Triana-Chavez, Universidad de Antioquia, Colombia Teresa Gárate, Instituto de Salud Carlos III, Spain

#### **\*Correspondence:**

Leidi Herrera, Laboratory of Parasite and Vector Biology, Institute of Tropical Zoology and Ecology, Science Faculty, Central University of Venezuela, AV. Los Ilustres-Antigua ETI, Caracas 1041A, Venezuela e-mail<EMAIL_ADDRESS>
Trypanosoma cruzi the etiological agent of American Trypanosomiasis or Chagas disease (ChD) is transmitted by triatomines vectors between mammals including man. T. cruzi has existed for circa 150 Ma in the Americas and nearly 10 million people are currently infected. The overlap between wild and domestic ecotopes where T. cruzi circulates is increasing. Host–parasite interactions have been determined by infection patterns in these cycles, all under natural or laboratorial conditions.This mini-review describes specific parasite niches, such as plant communities or biological corridors between domestic and wild landscapes, in order to help identify risk factors for ChD and define the boundaries between wild and domestic transmission cycles, with an emphasis on research undertaken in Venezuela.

**Keywords:Trypanosoma cruzi, domestic cycle, wild cycle, Chagas disease, Venezuela**

## **INTRODUCTION**

Parasites and their hosts form part of trophic webs and may be considered bioindicators of climate changes and anthropogenic impacts (1). American trypanosomiasis (AT) or Chagas disease (ChD) is a complex parasitosis caused by *Trypanosoma cruzi* (Kinetoplastida, Trypanosomatidae), which can be dispersed by enzootic or anthroponotic routes in trophic webs, which involve several mammals groups including human beings (**Figure 1**). So, this parasite affects currently, until 10 million people and as such can be considered a re-emerging public health problem especially in Venezuela (2, 3).

The *T. cruzi* life cycle begins when vectors (Hemiptera, Reduviidae, Triatominae) expel feces or urine with infective metacyclic trypomastigotes which then come into contact with mammals via intact mucous or skin abrasions. The trypomastigotes pass into the bloodstream and invade a wide range of tissues where they differentiate into amastigotes, epimastigote, and trypomastigotes once again. The latter are re-released into the bloodstream and can be imbibed by another or the same vector, which pass into the intestine and transform, once more, into metacyclic, performing a vectorial transmission (3).

Recent outbreaks of oral transmission in Brazil and other Latin American countries, including Venezuela, emphasize the importance of this alternative route in enzootic and zoonotic cycles (4).

*Trypanosoma cruzi* has been grouped into six discrete typing units (DTU): *T. cruzi* I (TcI) to *T. cruzi* VI (TcVI). The TcI– TcVI classification is, however, a relatively recent nomenclature and the associations of the different genotypes with particular hosts, ecotopes, or transmission cycles remains under debate (5).

*Trypanosoma cruzi* has existed in the Americas for *circa* 150 Ma and has been in contact with Amerindians for 15,000 years. The genome of this parasite in mummies from the American Pacific (7,500 BC to 1,500 AC) indicates a pre-Columbian origin thus breaking the myth of its establishment as a product of recent colonialism (6).

The host–parasite associations and risk factors associated with ChD is being described in recent studies of specific niches such as mammal caves, plant communities, and biological corridors between domestic and wild ecotopes, in order to widen the understanding of the boundaries between wild and domestic *T. cruzi* cycles in Venezuela.

## **VECTOR–PARASITE: PATTERNS OF WILD AND DOMESTIC TRYPANOSOMA CRUZI CYCLES**

Triatomines are eclectic in their ecological niches: they are found from 42°N to 46°S and between 400 and 1200 m.a.s.l. (7). A total of 140 species are grouped into 8 tribes and 15 genera. *Triatoma maculata*, *Rhodnius prolixus*, and *Panstrongylus geniculatus* are the most important in Venezuela by their frequency of infection by *T. cruzi* and their association with domestic and peridomestic ecotopes in economically depressed rural areas (8).

*Rhodnius prolixus* in Venezuela, is predominantly intradomiciliary with a high-reproduction rates, a voracious blood intake and fast defecation time, all of which are attributes of a primary vector. In the wild, this triatomine is predominantly found in palms with synanthropic vertebrates providing the blood source (9, 10). *T. maculata* is found in palms, dry trees, wooden fences, and bird nests near human dwellings. Its domiciliation, in function to phenotypic and genotypic discrimination according to its ecotopes, guarantee the previous consideration about its presence a risk factor for parasite transmission in Brazil, Colombia, and Venezuela (11–13).

Coconut palms (*Coccus nucifera*) is a suitable triatominae ecotope in peridomicile environments in north-eastern Venezuela, as was corroborated by the presence of 242 *R. prolixus* and 144 *T.*

*maculata* adults in 14 coconut palms just 5 m away from human habitations. PCR amplification of the D7 divergent domain of the 24S rRNA genes; the non-transcribed spacer of mini-exon genes and the size-variable domain of the 18S rRNA genes confirmed that 98% of the *R. prolixus* and 70% of the *T. maculata* individuals were infected by *T. cruzi*–TcI. Exploration of coconut and its derivatives in industry and ethno-botany could pose a risk by exposing human beings to contaminated triatomines fluids (14).

Other triatomines vector species can acts as boundaries between wild and domestic environments. Examples are *Eratyrus mucronatus* and *Panstrongylus rufotuberculatus* found in palms, tree holes, and the dens of animal reservoirs, in Bolivia, Colombia, and Venezuela (15, 16). *P. rufotuberculatus* is a widely dispersed triatomine; in Venezuela could be monitored in peridomiciles in Anzoátegui state when wildlife fauna was affected or natural enemies were altered. Inoculation of the intestinal content of these insects in murine model, shown invasion of chondral tissue, brain, and kidney, revealing novel clinical aspects to be considered in relation to ChD (16).

Other triatomine species is *P. geniculatus*, which has been associated to infection of rodents and marsupials in rural or domestic ecotopes. The loss of its natural niches has also promoted its avid penetration in human dwellings. This is particularly worrying as this insect has been imputed as parasite font in cases of oral transmission of ChD in Caracas, and other cities in Venezuela. The modification of reservoir niches by climate change or the human exploitation of landscapes favors its peridomestic and domestic colonization (8, 17, 18).

The recent report of K-DNA and satellite DNA of *T. cruzi* in the intestines of *P. geniculatus* from sites along the Orinoco River, near Amerindian settlements, was associated with records of this species in neighboring countries, which could constitute evidence of biological corridors of the parasite with potential impacts on indigenous populations (19).

## **RESERVOIR–PARASITE: THE INTERACTION OF WILD MAMMALS, HUMAN BEINGS, AND DOMESTIC ANIMALS**

Up until now, 180 species have been identified as reservoirs included in Artiodactyla, Carnivora, Cingulata, Chiroptera, Didelphimorphia, Lagomorpha, Perissodactyla, Pilosa, Primates (including man), and Rodentia orders (18).

*Trypanosoma cruzi* is considered as euryxenic according to the range of reservoirs it inhabits and eurytopic as regards the different organs it infects. Alternative transmission routes have also been reported in order to fluctuations in reservoir subpopulations, which could explain the plasticity of this zoonosis and urban outbreaks. The parasite may be orally transmitted via ingestion of infected triatomines, contaminated food, blood, or viscera from reservoirs (18, 20).

Studies of the distribution patterns of *T. cruzi* genotypes should consider ecological peculiarities since that genetic diversity has on the outcome of zoonosis or human disease (20). *T cruzi* Z3 in the southern Amazon (*Trichomys* rodent–*T. cruzi* complex in Brazil) and *T. cruzi* TcIII in the northern Amazon (*Dasypus*–*T. cruzi* complex in Venezuela) provide instances of the expansion of these wild genotypes into urban cycles (21, 22).

Particularly, *Dasypus novemcinctus* form part of an ancient enzootic *T. cruzi* cycle in the touristically important north-eastern region of Venezuela. These mammals act as TcIII reservoirs, as has been shown by the PCR amplification of the intergenic region of HSP60 genes for*T. cruzi* and the restriction digest of PCR products by *Eco*RV. The interaction of *Dasypus novemcinctus* with human beings, domestic animals, and peridomestic triatomines in this region, may be an important risk factor (22).

The ubiquity of *T. cruzi* in mammal reservoirs and its effect on host fitness represents an element that has been scarcely studied. Parasite isolates from *D. marsupialis*, *R. prolixus*, and *T. maculata* from rural and urban areas of Venezuela have yielded 105 flagellates/ml of blood in mice models, producing 80% mortality with neurological symptoms such as ataxia, paralysis, and sphincter relaxation. Alterations as meningo-encephalitis, edema of the neuropil and parasitism near vascular system could facilitate the hematological dispersion of the parasites. These neurological disorders could alter the behavior of mammals toward predators thus modifying parasite transmission in trophic web (23, 24).

## **CONCLUSION**

Parasitism implicates energy movement among organisms, affecting the interactions and robustness of some trophic webs. *T. cruzi* is a clonal parasite with wild and domestic cycles, some author has proposed that vector–mammal interaction and saturation vector feeding rates, depend on mammal density when the vector/mammal ratio is low and vector density when this ratio is high (25). The number of infected mammals is conditioned by their relative abundance, which thus influences their availability as a blood source for triatomines.

New incursion of some vectors or mammals reservoirs species in *T. cruzi* life cycle, is important in the epidemiology of AT and ChD. The potential trophic web can include ingestion of insects, contaminated food, or host carnivorous behavior, which could be the primary route for *T. cruzi* transmission in some wild cycles (**Figure 1**). Synanthropic mammals and vectors are not excluded from this, thus, providing a way by which the wild and domestic cycles could be crossed (26–28).

### **ACKNOWLEDGMENTS**

The author would like to thank Luis Villamizar for laboratory assistance and Elizabeth Ferrer and Daisy Lozano for help with manuscript preparation. Funding was provided by the: CDCH (Project PG-03-8171-2011); P.E. FONACIT (No<PHONE_NUMBER>); Misión Ciencia (Project No.<PHONE_NUMBER>-6).

#### **REFERENCES**

and its surroundings (Piauí, Brazil), endemic for Chagas disease. *Trans R Soc Trop Med Hyg* (2005) **99**:379–88. doi:10.1016/j.trstmh.2004.07.006

28. Kribs-Zaleta CM. Estimating contact process saturation in sylvatic transmission of *Trypanosoma cruzi* in the United States. *PLoS Negl Trop Dis* (2010) **4**(e):656. doi:10.1371/journal.pntd.0000656

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The Guest Associate Editor Juan-Carlos Navarro declares that, despite being affiliated to the same institution as the author Leidi Herrera, the review process was handled objectively and no conflict of interest exists.

*Received: 17 July 2014; accepted: 10 November 2014; published online: 28 November 2014.*

*Citation: Herrera L (2014) Trypanosoma cruzi, the causal agent of Chagas disease: boundaries between wild and domestic cycles in Venezuela. Front. Public Health 2:259. doi: 10.3389/fpubh.2014.00259*

*This article was submitted to Epidemiology, a section of the journal Frontiers in Public Health.*

*Copyright © 2014 Herrera. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Ultrastructural study on tissue alterations caused by trypanosomatids in experimental murine infections

## **Héctor J. Finol \* and Antonio Roschman-González**

Center for Electron Microscopy, Faculty of Sciences, Central University of Venezuela, Caracas, Venezuela

target for trypanosomatids.

#### **Edited by:**

Rubén Bueno-Marí, University of Valencia, Spain

#### **Reviewed by:**

Charles E. White, Charles E. White's Biostatistical Consulting, LLC, USA Hyacinth Idu Hyacinth, Medical University of South Carolina, USA

#### **\*Correspondence:**

Héctor J. Finol, Center for Electron Microscopy, Faculty of Sciences, Central University of Venezuela, Apartado 40.494, Los Chaguaramos, Caracas 1041A, Venezuela e-mail<EMAIL_ADDRESS>
## **INTRODUCTION**

At the present time, there is a vast literature concerning the effects of protozoan parasites in the ultrastructure of mammalian tissues. These works include horse and mouse skeletal muscles infected by *Trypanosoma evansi* (1, 2) and *Toxoplasma gondii* (3, 4), human skeletal and mouse cardiac muscles by *Trypanosoma cruzi* (5, 6), avian skeletal muscle by *Plasmodium cathemerium* (7), and human skeletal muscle by *Plasmodium falciparum* (8). Alterations in the mouse adrenal gland and liver were provoked by *Trypanosoma evansi* (9, 10) and *Plasmodium berghei* (11, 12). In this context, it would be interesting to know if structural changes observed are similar in all studied species. Furthermore, with this investigation we intend to perform a systematic work on the ultrastructure of alterations in experimental murine infections by some trypanosomatids. This could help to understand better the biology of trypanosomatids in vertebrate host.

## **MATERIALS AND METHODS**

## **EXPERIMENTAL INFECTIONS**

For experimental infections, Balb/c mice were used. They were divided into 3 groups of 10 mice each one for the three used species (*Trypanosoma cruzi*, *Trypanosoma evansi*, and *Leishmania mexicana*), and one additional 10-mice group was used as a control. Animals were infected by intraperitoneal (i.p.) route with inocula consisting of 10 parasites/g of animal body weight and uninfected mice were kept as controls. Three mice from each group were randomly selected after prepatent period and killed under anesthesia during peaks of infection. Then tissue samples were removed and processed for transmission electron microscopy. The experimental procedures were approved by the ethical committee of the Sciences Faculty at the Central University of Venezuela, and the work was conducted in agreement with the regulatory standards.

## **TRANSMISSION ELECTRON MICROSCOPIC STUDY**

The ultrastructural study in different tissues of mice experimentally infected with isolates of Trypanosoma evansi,Trypanosoma cruzi, and Leishmania mexicana reveals changes in cardiac myocytes, skeletal muscle fibers, and hepatic, adrenal, kidney, and spleen cells. Some of these changes were cytoarchitectural and others consisted of necrosis. Alterations in the microvasculature were also found.The mononuclear cell infiltrate included neutrophils, eosinophils, and macrophages. This work shows that diverse mice tissues are important

> Tissue samples were fixed with Karnovsky's solution, in phosphate buffer at pH 7.4 and 320 mOsm, post-fixed in 1% OsO4, and embedded in epon resin. Sections were cut with a diamond knife in a Porter-Blum MT-2B ultramicrotome and stained with uranyl acetate and lead citrate. Ultrathin sections were observed in a Jeol JEM – 1011 transmission electron microscope, at an accelerating voltage of 80 kV.

## **RESULTS**

**Keywords: pathology, ultrastructure, murine tissues, experimental infections, trypanosomatids**