Efficacy of a topical combination of fipronil-permethrin against Rhodnius prolixus on dogs.

A controlled clinical trial was carried out to assess the mortality and repellency of a new topical combination of fipronil-permethrin (Effitix® Virbac, Mexico) against Rhodnius prolixus in dogs. Ten medium-size dogs (10-15 kg) with short hair were used. The dogs were exposed to 8 adult triatomines once weekly for 7 weeks. On the control day (D0), the dogs were exposed to the insects without treatment. On D7, the dogs were immediately treated with a spot-on 2.2 ml pipette containing 134 mg of fipronil and 1200 mg permethrin after exposure to the insects. The dose was repeated after 4 weeks following the manufacturer's instructions. Repellency at D0 was, 0 % and the insects had a high blood content. After 12 h post-contact, repellency was 86.3 % and slowly decrease though D21 and D28. On D7, none of the insects survived after 3 h of feeding on the treated dogs. On D14, D35 and D42, all insects died within 12 h post-feeding, whereas no mortality was observed in the control D0 (P < 0.05). The results of this study indicated that administration of the product following the manufacturer's instructions was efficacious at inducing rapid mortality of R. prolixus and therefore could be useful to prevent the transmission of American trypanosomiasis in dogs.

Protective immunogenicity of the paraflagellar rod protein 2 of Leishmania mexicana.

Paraflagellar rod proteins (PFR) are a potent immunogen against experimental Trypanosoma cruzi infection. PFR are highly conserved among kinetoplastid parasites. We therefore evaluated the immunogenicity of the Leishmania mexicana pfr-2 gene and protein product in the hamster model of American cutaneous leishmaniasis. Immunization with pfr-2 DNA-induced specific antibody, confirming immunogenicity. Subsequent challenge with 10,000 and 500 stationary phase L. mexicana promastigotes respectively, resulted in delayed appearance of lesions, and significant reduction in lesions post infection in male hamsters, yet exacerbated lesions in female hamsters. Immunization with recombinant PFR-2 protein (rPFR-2) prevented lesion development in female hamsters challenged with L. panamensis, but was ineffective against L. mexicana. Nevertheless, prime boost immunization of female hamsters with rPFR and pfr-2 DNA significantly reduced lesion size following challenge with 500 L. mexicana promastigotes, supporting the relevance of PFR-2 as a potential vaccine constituent.

Spatial and temporal patterns of phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in northern Argentina.

Phlebotomine sand flies (Diptera: Psychodidae) were captured in an area of Argentina endemic for American cutaneous leishmaniasis (ACL). A total of 44,944 flies were collected during a 130-wk interepidemic period from 1990 through 1993. These sand flies included Lutzomyia neivai (Pinto) (97.8%), Lutzomyia migonei (Franca) (1.2%), Lutzomyia cortelezzii (Brèthes) (0.8%), Lutzomyia shannoni (Dyar) (0.1%), and Lutzomyia punctigeniculata (Floch and Abonnenc) (0.1%). Lutzomyia neivai was more abundant in secondary forests and peridomestic environments associated with human cases than in primary forest or xeric thorn scrub areas. Time series analyses of species densities suggested a bimodal or trimodal annual pattern related to rainfall peaks, a 5-wk reproductive cycle, and peridomestic local populations that were located adjacent to secondary forests. In general, sand fly abundance was correlated with the rainfall of the previous year. Lutzomyia neivai spatial distributions were consistent with ACL incidence patterns during the study and in the recent outbreaks in Argentina. However, Lu. migonei also may be involved in peridomestic transmission. Our results suggest that there is a need for improved, long-term surveillance of sand flies and ACL cases, as well as development of effective intervention strategies.

Comparative vectorial efficiency of Lutzomyia evansi and Lu. longipalpis for transmitting Leishmania chagasi.

The infection rates and development of Leishmania chagasi in two sandfly species, Lutzomyia evansi and Lutzomyia longipalpis, were evaluated under natural and experimental conditions. Natural infection rates of Lu. evansi in San Andrés de Sotavento (Colombia) and Montañas de Peraza (Venezuela) (0.05 and 0.2%, respectively) were similar to those previously recorded for this species in Colombia and Venezuela and for Lu. longipalpis in many foci of American Visceral Leishmaniasis (AVL). Both sand fly species were able to support the development of two Colombian strains of L. chagasi experimentally acquired from dogs, hamsters or membrane feeders. However, the experimental infection rates and the sequence of parasite development in the guts of these sand flies revealed that parasite colonisation, differentiation, migration and attachment were more frequent and uniform in Lu. longipalpis than in Lu. evansi. This is consistent with a more recent association between L. chagasi and Lu. evansi, and these results might help to explain the irregularity of AVL outbreaks in foci where Lu. evansi has been reported as the sole vector.

Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi.

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low ((1/2)0-(1/4)8 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.

Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15 percent of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low (1/20-1/48 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs

Canine visceral leishmaniasis: dog infectivity to sand flies from non-endemic areas.

Canine visceral leishmaniasis (VL), caused by Leishmania infantum (Leishmania chagasi in the New World), is a zoonotic, endemic disease in Western Europe and Latin America. The potential spreading to new regions was suggested by the appearance of canine VL among foxhounds in the US. Although the sand fly vectors in the major foci of transmission have been described, no information exists on other sand flies that could propagate the infection outside endemic areas. We evaluated the capacity of Lutzomyia shannoni (Dyar) and Lutomyia youngi (Feliciangeli & Murillo), which are widely distributed in the New World, to acquire L chagasi (Cunha and Chagas) infections. A high proportion of L youngi were infected after feeding on an oligosymptomatic dog (51 per cent) or a polysymptomatic individual (95 per cent), but the intensity of infection was low (< 200 promastigotes/fly). L shannoni became infected only by feeding on the polysymptomatic dog, and the infection rate was lower (9 per cent) than in Lutzomyia longipalpis (36 per cent), and Lutzomyia evansi (Nunez-Tovar) (Lutz and Neiva) (38 per cent), but the intensity of infection (200 to > 500 promastigotes/fly) was comparable (L longipalpis) or higher (L evansi) than in the New World vectors. It is hypothesised that the presence of infected dogs in areas where L shannoni or L youngi occur could initiate new endemic cycles of VL in both South and North America.

Canine visceral leishmaniasis in Colombia: relationship between clinical and parasitologic status and infectivity for sand flies.

We studied the reservoir competency of canines with distinct clinical presentations of Leishmania chagasi infection. The parasitologic status of asymptomatic and symptomatic dogs was determined by standard culture methods Infectivity was assessed by multiple xenodiagnoses with Lutzomyia longipalpis, over a period of 2-11 months. Asymptomatic dogs were non-infective (0 of 5) while 2 of 7 oligosymptomatic dogs infected L longipalpis, transmitting the parasites at low rates (range 0.9-5.2% of engorged flies). Polysymptomatic dogs transmitted L. chagasi more frequently (4 of 8 dogs) and reached higher infection rates (range 5.0-22.5% of engorged flies). The skin of the ear tended to be more infective to sand flies than that of the abdomen. Polymerase chain reaction hybridization (PCR-H) was a sensitive method for detection of L. chagasi, yielding the highest positive rate in serum (16 of 17 dogs) with no distinction between clinical groups. No association between skin positivity by PCR-H and infectivity to sand flies was found. The infectivity of dogs from clinically comparable groups from Colombian and Mediterranean foci differed. This may be a reflection of varied nutritional conditions or vector competency of distinct sand fly species.

Association of Lutzomyia columbiana (Diptera: Psychodidae) with a leishmaniasis focus in Colombia due to species of the Leishmania mexicana complex.

In Colombia, Leishmania mexicana has a scattered geographical distribution and no sand fly vectors have been associated with its transmission. During the present study, the anthropophilic sand fly Lutzomyia columbiana was found to be the only species collected using diverse methods, in a small focus of Le. mexicana in the municipality of Samaniego, SW Colombia. Ecological data indicate that this sand fly species is present in both peri and intradomestic habitats, where it readily bites man. Further evidence comes from experimental infections of wild-caught Lu. columbiana with Le. mexicana after feeding on infected hamsters. Based on these results, it is suggested that this sand fly is the most likely vector in the study area, suggesting the existence of a previously unknown sand fly-parasite association.

Dynamics of Leishmania chagasi infection in small mammals of the undisturbed and degraded tropical dry forests of northern Colombia.

The infection rate with Leishmania chagasi and the population dynamics of small mammals were studied in an undisturbed forest reserve (Colosó) and an area of highly degraded forest (San Andrés de Sotavento [SAS]) in northern Colombia, both endemic for visceral leishmaniasis. Live trapping of mammals was done every month, and species, age, sex and reproductive status determined. L. chagasi was detected in samples of skin or spleen by the polymerase chain reaction, after extraction of deoxyribonucleic acid using specific primers (DB8/AJS3), and dot blood hybridization. Didelphis marsupialis was found to be infected in Colosó (3/21, 14.3%) and SAS (13/137, 9.5%); its relative abundance was higher in SAS (93/113, 82% of the captures). Although Proechimys canicollis was also found to be infected in Colosó (3/34, 8.8%) and SAS (2/4), its relative abundance was much lower (4%) in SAS than in Colosó (56% of 77 animals captured). Sciurus granatensis, Marmosa robinsoni, Heteromys anomalus, Zygodontomys brevicauda and Metachirus nudicaudatus were less common, and no L. chagasi infection was detected in them.

Reinfection in American cutaneous leishmaniasis: evaluation of clinical outcomes in the hamster model.

There is no clear understanding of the outcome of reinfection in New World cutaneous leishmaniasis, and its role in the relationship to the development of protection or secondary disease. For this reason, reinfection experiments with homologous (Leishmania panamensis-L. panamensis) and heterologous (L. major-L. panamensis) species of leishmaniae were conducted in the hamster model. The different protocols for primary infections prior to the challenge with L. panamensis were as follows: (a) L. major, single promastigote injection, (b) L. major, three booster infections, (c) L. panamensis, followed by antimonial treatment to achieve subclinical infection, (d) L. panamensis, with active lesions, (by antimonial treatment to achieve subclinical infection, (d) L. panamensis, with active lesions, (e) sham infected, naive controls. Although all reinfected hamsters developed lesions upon challenge, animals with active primary lesions due to L. panamensis, and receiving booster infections of L. major had the most benign secondary lesions (58-91% and 69-76% smaller than controls, respectively, P < 0.05). Subclinically infected animals had intermediate lesions (40-64% smaller than controls, P < 0.05), while hamsters which received a single dose of L. major had no significant improvement over controls. Our results suggested that L. major could elicit a cross protective response to L. panamensis, and that the presence and number of amastigotes persisting after a primary infection may influence the clinical outcome of reinfections.

Reinfection in American cutaneous leishmaniasis: evaluation of clinical outcomes in the hamster model

There is no clear understanding of the outcome of reinfection in New World cutaneous leishmaniasis, and its role in the relationship to the development of protection or secondary disease. For this reason, reinfection experiments with homologous (Leishmania panamensis-L. panamensis) and heterologous (L. major-L. panamensis) species of leishmaniae were conducted in the hamster model. The different protocols for primary infection prior to the challenge with L. panamensis were as follows: (a) L. major, single promastigote injection, (b) L. major, three booster infections, (c) L. panamensis, followed by antimonial treatment to achieve subclinical infection, (d) L. panamensis, with active lesions, (e) sham infected, naive controls. Although all reinfected hamsters developed lesions upon challenge, animals with active primary lesions due to L. panamensis, and receiving booster infections of L. major had the most benign secondary lesions (58-91 per cent and 69-76 per cent smaller than controls, respectively, P<0.05). Subclinically infected animals had intermediate lesions (40-64 per cent smaller than controls, P<0.05), while hamsters which received a single dose of L. major had no significant improvement over controls. Our results suggested that L. major could elicit a cross protective response to L. panamensis, and that the presence and number of amastigotes persisting after a primary infection may influence the clinical outcome of reinfections.

The inflammatory response promotes cutaneous metastasis in hamsters infected with Leishmania (Viannia) panamensis.

The influence of nonspecific and immunologically elicited inflammatory responses on the development of metastatic lesions was examined in the hamster model of Leishmania (Viannia) panamensis infection. Delayed type hypersensitivity (DTH) responses were induced using the contact sensitizing agent DNFB (2, 4-dinitro-1-fluorobenzene) and infection with L. panamensis followed by intradermal application of leishmanin. Nonspecific inflammatory response was achieved by the surgical excision of toes. The inductive and eliciting procedures were performed on the ears and fore and hind paws of the right side of experimental groups of hamsters that were inoculated in the snout with a highly metastatic strain of L. panamensis (MHOM/COL/84/1099). Skin metastases were detected by physical evaluation at 15-day intervals over a period of 7-8 mo. Suspected metastases were parasitologically confirmed by culture of tissue fluid aspirated from the lesion. The frequency of metastatic lesions was greater in hamsters subjected to inflammatory stimuli (14/38) than control animals (6/33; P = 0.035). Likewise, the frequency of metastases at the site of induction and elicitation of inflammation (18/22 lesions) in the experimental groups was greater than that observed at the same site in control animals (5/11 lesions; P = 0.017). These findings support a causal relationship between inflammatory response and the development of lesions in this model of secondary disease caused by L. panamensis.

Bionomics of Lutzomyia evansi (Diptera: Psychodidae) vector of visceral leishmaniasis in northern Columbia.

The feeding behavior, seasonality, and natural infection rate of Lutzomyia evansi (Nuñez-Tovar) with Leishmania chagasi (Cuna & Chagas) was studied during a 12-mo period at 2 hamlets, El Contento and Vidales. Sand fly abundance in extra-, peri-, and intradomestic habitats was evaluated with sticky traps and CDC light traps, whereas human bait and Shannon trap collections were made only in peridomestic habitats. All trapping methods showed a clear predominance of L. evansi throughout the year. Sand flies were present during most of the year, with the exception of the driest months (February and March). Although the total number of sand flies was higher in El Contento than in Vidales, a larger proportion of L. evansi was found in intradomestic habitat than in the peri- and extradomestic habitats at Vidales. Also, sand flies from Vidales had a higher infection rate with L. chagasi than did those from El Contento. Although 2 of 9 promastigote infections detected in L. evansi were identified as L. chagasi, the difficulty of isolating and propagating leishmania strains from this visceral leishmaniasis focus precluded characterization of most parasite samples. Parous and infected sand flies were most abundant toward the end of the rainy season (October-December). For this reason, control strategies based on reducing sand fly populations or avoiding human-vector contact should be concentrated during the October-December period.

Infectivity of Trypanosoma rangeli in a promonocytic mammalian cell line.

The existence of an intracellular stage of Trypanosoma rangeli in the vertebrate host was evaluated by experimental infection of the U937 histiocytic cell line with the San Agustín strain and the Ub66-5b clone. The identity of the parasites at the beginning and end of the experiments was confirmed through biological behavior in the vector and mammal hosts, isoenzymes, polymerase chain reaction (PCR), and monoclonal antibodies. Infectivity to U937 cells of T. rangeli obtained from culture and salivary glands was evaluated under different experimental conditions. These included 34 C vs. 37 C, opsonized vs. nonopsonized parasites, and 2, 4, 6, 24, 48, and 72 hr of cell-parasite contact. Trypanosoma rangeli adopted a characteristic nondividing amastigote-like form within U937 cells, which was different in size (P = 0.001) from Trypanosoma cruzi amastigotes. Culture forms of T. rangeli were more infective than parasites from salivary glands (P = 0.049) but were less infective than T. cruzi (P = 0.0001). Variations in temperature (34-37 C) and complement opsonization did not affect infectivity. Viability of intracellular forms was confirmed by feeding Rhodnius prolixus with T. rangeli-infected cells. Resistance of T. rangeli to the intracellular milieu could be an important mechanism in producing chronic infections in mammals and in the infection of triatomines.

Phlebotomine sandflies associated with a focus of cutaneous leishmaniasis in Valle del Cauca, Colombia.

A survey was made of the phlebotomine sandfly fauna of La Guaira, a village with coffee plantations near Cali, Colombia, from which cases of American cutaneous leishmaniasis had been reported due to Leishmania (Viannia) panamensis and Le. (V.) braziliensis. Among six species of sandfly collected on human bait, Lutzomyia youngi was most important in terms of biting nuisance. Lu.columbiana, Lu.lichyi and Lu.scorzai as well as Lu.youngi adults occurred throughout the year. Sandfly man-biting activity occurred throughout the night and was highest within 2 h of sunset. Despite its abundance in nocturnal samples, Lu.youngi was rarely taken in diurnal resting site collections. In contrast, Lu.lichyi was collected on tree-trunks during the day in large numbers and was the only species biting in daylight. The implications of these and other findings for leishmaniasis control measures in La Guaira are considered.

Evaluation of deltamethrin-impregnated bednets and curtains against phlebotomine sandflies in Valle del Cauca, Colombia.

The effectiveness of bednets and curtains (nylon mesh 64 per cm2) impregnated with deltamethrin at 26 mg a.i./m2 in reducing the biting nuisance caused by three phlebotomine sandfly species: Lutzomyia columbiana, Lu.lichyi and the predominant Lu.youngi (Diptera: Psychodidae), was evaluated at La Guaira, a rural settlement in Valle de Cauca near Cali, Colombia. Pairs of volunteers collected sandflies under impregnated bednets, in rooms protected by impregnated curtains or in unprotected rooms in a randomized matched design. Collections were made in three houses per night on three consecutive nights, so that each house was sampled under each of the three treatments. This routine was repeated at 2-week intervals for 6 months. There was no significant difference between the overall numbers of sandflies collected in rooms with or without impregnated curtains. Only 0.14 sandflies/man-hour were caught on human bait under impregnated bednets, significantly fewer than the numbers collected on human bait outside the nets in the same room (1.91) or in unprotected rooms (3.29). In a second set of experiments carried out in La Guaira and the neighbouring community of Jiguales, the effect of deltamethrin impregnation was evaluated by comparing numbers of sandflies collected on human bait under treated and untreated nets. Significantly fewer were collected under the impregnated nets (0.25 v. 0.69/man-hour). Wild-caught female Lu.youngi exposed to treated netting for 2 min in the laboratory all died with 24 h. The impact of deltamethrin-impregnated bednets was considered to be useful against Lu.youngi and other potential vectors of leishmaniasis in such communities.

An attempt to control Phlebotomine sand flies (Diptera: Psychodidae) by residual spraying with deltamethrin in a Colombian village

An attempt was made to control phlebotomine sand flies biting indoors in a rural community near Cali, Colombia, using the residual insecticide "K-Othrine" (deltamethrin) sprayed on the inside walls of houses. Twelve houses were divided into matched pairs based on physical characteristics, one house in each pair being left untreated while the inside walls of the other were sprayed with 1 deltamethrin at a concentration of 500 mg a.i./m2. Sand flies were sampled each week using protected human bait and sticky trap collections for four months after spraying. The number of sand flies (Lutzomyia youngi) collected on sticky traps was significantly lower (P = 0.004) in the untreated houses than in the treated ones with which they were matched. This difference was not significant for L. columbiana; the other anthropophilic species were not present in large numbers. The numbers collected on human bait in treated and untreated houses were not significantly different for either species. Activity of the insecticide as determined by contact bioassays remained high throughout the study and failure to control the insects was attributed to two factors: the tendency of sand flies to bite before making contact with the insecticide and the fact that the number of sand flies that entered houses represented a relatively small proportion of the population in the wooded areas surrounding the settlement in the study.