A canine model of experimental infection with Leishmania (L.) mexicana
© Cruz-Chan et al.; licensee BioMed Central Ltd. 2014
Received: 29 January 2014
Accepted: 3 August 2014
Published: 9 August 2014
Cutaneous leishmaniasis is a tropical disease affecting over one million patients annually and Leishmania (L.) mexicana is one of the major etiological agents in the Americas. Here we established the first experimental infection of L. (L.) mexicana in canids.
Beagle dogs were infected intradermally with culture-derived L. (L.) mexicana. We followed skin ulcer development, histopathological signs, parasite burden and the immune status of the infected dogs.
All infected dogs developed uniform oval-craterform ulcers similar to those observed in humans, associated with mixed T helper 1/T helper 2 immune responses. Parasites were detected in the healed lesions 15 weeks post-infection. Higher anti-Leishmania IgG levels correlated with larger lesions and high IgG1/IgG2 ratio was associated with some level of splenomegaly.
The canine model described in this work will be of use for further understanding of L. (L.) mexicana immunopathogenensis, and for drug and vaccine development.
KeywordsLeishmaniasis Canine model Experimental infection
The leishmaniases are a group of parasitic disease affecting up to 1.4 millions people annually . Cutaneous (Chiclero’s Ulcer), mucocutaneous and visceral leishmaniasis (Kala-azar) are the different clinical forms of the disease, which is caused by parasites from two subgenus: Leishmania (Leishmania) and Leishmania (Viannia). The parasite is transmitted by sand flies of the Phlebotomus and Lutzomyia genus in the old and new world, respectively. Cutaneous leishmaniasis (CL) ranks ninth of infectious neglected-tropical diseases, with an estimated burden of 1.2 million new cases per year, and of 770,000 DALYs (disability-adjusted life years) [1–3]. L. (L.) mexicana is one of the major species responsible for CL in the Americas. It can be found in Argentina, Brazil, Costa Rica, Guatemala, and as far north as Mexico [1, 4, 5]. In Mexico, the Ministry of Health reports an incidence of 500-900 cases annually, although the disease is likely to be under-reported [5, 6]. Drug treatment for infected patients is complicated, with only few drugs available with limited efficacy, and a vaccine is still in early experimental stages [1, 5, 7, 8].
Dogs are believed to play an important role as a domestic reservoir of Leishmania parasites, as demonstrated for L. (L.) chagasi. This is due to relatively high natural infection rates in many regions, ranging from 4 to 60% [10–15] and the life-long persistence of parasites, particularly in the skin, with or without clinical signs of disease . Drug treatment of infected dogs is not recommended, to prevent the emergence of drug resistance of the parasite . As a consequence, some countries such as Brazil are recommending the culling of infected dogs to prevent spreading of the disease, although the efficacy of such measure has been criticized . An important priority is thus to develop veterinary vaccines, which require appropriate animal models for their evaluation. Rodent models have been extensively used for the study of Leishmania infections, including inbred and outbred mouse strains and hamsters [18, 19], and have provided key information on the immunopathology of the disease. However, studies in dogs are also warranted for a further understanding of their role in Leishmania transmission and for testing drug and vaccine efficacy [20, 21].
Most studies on canine leishmaniasis have focused on the visceral form caused by L. (L.) chagasi or L. (L.) infantum, and are based on observations of both naturally and experimentally infected animals [21–27]. Unexpectedly, canine cutaneous leishmaniasis has been little studied in domestic dogs and is poorly described. A high prevalence of infection by L. (V.) braziliensis, L. (V.) guayanensis, and L. (L.) panamensis has been reported in dogs from Colombia [27, 28]. In Mexico, cases of canine leishmaniasis have been reported, possibly caused by L. (L.) infantum, although the parasite species was not determined. Cases of natural infection with L. (L.) mexicana have also been described . Dogs naturally infected with L. (V.) braziliensis were found to present thrombocytopenia, anemia and skin lesions . Parasites could be observed in 35% of the lesions, which also presented diffuse chronic inflammation of the dermis and fibrinoid degeneration, and less frequently some vasculitis . However, experimental models of canine cutaneous leishmaniasis are scarce. The experimental infection with L. (V.) brasiliensis was found to induce a cutaneous lesion in three of four mongrel dogs, 4-8 months after infection, and the lesions tended to heal after 3-5 months . Histologically, these lesions had similar characteristics as those observed in naturally infected animals , but additional studies are needed to further characterize this dog model.
In this study, we developed a canine model of cutaneous leishmaniasis caused by experimental infection with L. (L.) mexicana parasites, the main species circulating in Southern Mexico and Central America. We described the clinical, parasitological and immunological aspects of the experimental infection in Beagle dogs, which provide a good model for the future testing of novel drugs or vaccines against cutaneous leishmaniasis, as well as for further studies on the immunopathogenensis of this canine host.
Six Beagle dogs from three litters and aged between two and three months were used. Animals were acclimated for three/four months in the animal facility. Temperature, light and food were controlled. They received treatment against helminths and were vaccinated against Rabies virus, Canine distemper virus, Type 2 Adenovirus, Coronavirus, Parainfluenza, Parvovirus and Leptospira. All animal procedures were performed according to national and international guidelines approved by the Institutional Bioethics Committee from the Autonomous University of Yucatan (authorization number CBI-CIR-11-04).
Parasites and infection
Leishmania (L.) mexicana MHET/MX/97/Hd18 strain was cultured in 199 media with 15% of fetal bovine serum, 5 μM mercaptoethanol, 20 mM sodium pyruvate, 100 IU penicillin and 100 mg/mL streptomycin, and 5% of filtered human urine. Under general anesthesia (ketamine/xilazine, 8:1 mg/kg, iv), Beagle dogs received 7 × 107 promastigotes resuspended in 50 μl of PBS solution intradermally on the shaved back .
Beagle dogs were under daily examination for clinical signs including rectal temperature, breath and heart rate. Additionally, feces color, presence of polydipsia, polyuria and/or any abnormal excretions were observed. Skin lesions were photographed and measured weekly using a Vernier Caliper over a period of 15 weeks.
SYBR Green-based real time-PCR was optimized to quantify L. (L.) mexicana parasite burden in prescapular lymph nodes and skin lesions. First, 25 mg of tissue was ground with a morter and pestle and DNA was obtained with a Wizard® Genomic DNA purification Kit (Promega Madison WI) following the manufacturer’s instructions.
We used primers targeting a 140 bp sequence from L. (L.) mexicana minicircle: forward primer: 5′-AATGCGAGTGTTGCCCTTTTG-3′ and reverse primer: 5′-GCCGAACAACGCCATATTAACC-3′ . Reactions contained 50 ng of DNA in a reaction volume of 20 μl with 500 nM (each) forward and reverse primers, and 1× Kappa SYBR FAST universal qPCR mix. Reactions consisted of a 10 min activation at 95°C followed by 40 cycles of 15 s at 95°C and 1 min 60°C, and a high resolution melt curve analysis at the end of the reaction. A standard curve was prepared with uninfected dog DNA spiked with serial dilutions of L. (L.) mexicana DNA covering a dynamic range of 150 to 1.5 × 106 parasite equivalent/reaction. All samples and standards were run in triplicates and the standard deviation among triplicates was less than 0.6 Cq.
Dogs were euthanized 15 weeks post-infection with a barbiturate overdose and necropsies were performed immediately registering detail of cavities, organs and fluids. Spleen and Liver were extracted, weighted and measured . Biopsies from the skin, spleen, prescapular lymph node and liver tissues were embedded in paraffin and sections were stained with hematoxylin and eosin to evaluate lesions and/or parasites.
The humoral immune response induced by L. (L.) mexicana infection was evaluated by measuring levels of total IgG, IgG1 and IgG2 subtypes, against parasite lysate and NH36 recombinant antigen [37, 38]. Soluble L. (L.) mexicana Antigen (SLA) was prepared from parasites cultured as described previously following 6 days culture in 199 Hanks® medium (Life technologies Carlsbad CA). Briefly, 4 × 105/mL parasites were washed three times with phosphate buffer solution (PBS) pH 7.2 and sonicated by three ultrasound cycles (Sonics Vibra Cell 130, Newtown CA) of 1 min at 45 W at 4°C. The lysed suspension was centrifuged at 5000 × g for 30 min at 4°C. The supernatant was recollected and stored in aliquots at 70°C until used . Recombinant NH36 (rNH36) was generously provided by Dr. Peter Hotez (Baylor College of Medicine, Houston, TX, USA). Ninety six-well plates were coated overnight with soluble L. (L.) mexicana MHET/MX/97/Hd18 antigen (20 μg/ml) or rNH36 recombinant protein (2.5 μg/ml). After blocking, plasma samples (1:100 and 1:50 dilutions for total IgG and IgG isotypes, respectively) were incubated for 1 h at 37°C, and then washed. Phosphatase-conjugated rabbit anti-dog IgG (1:4000 dilution, Sigma, USA), peroxidase-conjugated goat anti-dog IgG1 or sheep anti-dog IgG2 (1:1000 dilution, Serotec, UK) were added and after washing, the plates were revealed with pNPP or o-phenylenediamine substrates, respectively, and absorbance was measured on a Bio-Rad 550 microplate reader.
Cytokines IFNγ and IL-10 levels were quantified in plasma as well as spleen and liver extract supernatants. For this, 1 g of fresh tissue was ground with a mortar and pestle and resuspended with 2 mL of RPMI-1640 medium (Sigma-Aldrich Inc, St. Louis, MO). The homogenates were centrifuged at 10,000 g for 15 minutes at 4°C and the supernatants were stored at -70°C . IFNγ and IL-10 DuoSet Canine kits (R&D Systems Inc., Minneapolis) were used to measure cytokine levels in tissue supernantants and plasma samples according to the instructions of the manufacturer.
Pathology and histopathology
Analysis of antibody isotypes showed a gradual IgG isotype switching over the course of infection, from a predominant IgG2 isotype at four weeks post-infection to a predominant IgG1 isotype at 12 weeks post-infection, as indicated by the changes in IgG1/IgG2 ratio (Figure 5B). A high IgG1/IgG2 ratio at 12 weeks post-infection was significantly associated with splenomegaly (R2 = 0.68, P = 0.04), and tended to be associated with an increased parasite burden in the lesion (R2 = 0.42, P = 0.15). We also evaluated NH36-specific antibodies. Anti-NH36 IgG levels reached their maximum at 8 weeks post-infection, but then presented some decrease at 12 weeks post infection (Figure 5C).
While visceral leishmaniasis has been extensively studied in dogs and several experimental models have been described [20, 26, 34, 41–43], very little is known about canine cutaneous leishmaniasis. We present here a first detailed model of experimental infection with L. (L.) mexicana in Beagle dogs.
Infection of dogs with L. (L.) mexicana parasites led to the development of a typical ulcerated skin lesion in all six infected animals, with a rather rapid onset after 3-4 weeks of infection. Thus, in spite of some variability in terms of lesion size and duration, this infection model appeared very reproducible. A previous attempt of infection of mongrel dogs with 3 × 107 L. (V.) braziliensis promastigotes resulted in cutaneous lesion development in only three of four animals, and after a longer and variable incubation period of 8-32 weeks . Lesion duration in the L. (V.) braziliensis model was also highly variable, ranging from 16 to 32 weeks . This elevated variability and time length made this former model of limited practical use. A higher parasite dose (1 × 109) has also been used in a model of canine visceral leshmaniasis, but it led to limited clinical signs .
The clinical and pathological evolution of L. (L.) mexicana we described in this study was also very similar to what has been described for naturally-occurring cutaneous leishmaniasis in dogs. Thus, dogs naturally infected with L. (V.) braziliensis or L. panamensis present skin lesion varying in size from 0.4-10 cm [27, 29]. Similarly, the histopathologic damage that we observed in the skin and other tissues is very similar to that observed in naturally infected animals [32, 45]. Remarkably, our observation of lymphoid infiltration in the kidney is a first report indicating possible renal damage in experimental cutaneous leishmaniasis. The induction of glomerulonephritis during infection with L. (L.) infantum has been recently attributed to the deposition of immune complex following a limited cellular immunity . In addition, it is important to note that L. (L.) mexicana can cause a rather wide spectrum of clinical disease, including diffuse and visceral forms in some cases, particularly in patients with low immune status [47–49].
Also similar to the natural infection is the apparent absence of parasites in the healed lesion as assessed microscopically in tissue sections . However, using a technique with higher sensitivity such as qPCR, we clearly demonstrated an important parasite burden up to 15 weeks after infection, both in the skin and in the secondary lymphoid organs of all six infected animals. While this assay relies on the detection of parasite DNA, the presence of live parasites is highly likely, as observed in many animal models of Leishmania infection.
These important results suggest that infected dogs have reached an asymptomatic stage, but parasite persistence indicated that, on one hand infection may be reactivated upon alteration of their immune status, and on the other hand, they remain a potential source of infection and may serve as a reservoir of the parasite. In Brazil, it has been clearly established that domestic dogs are the main reservoir of L. chagasi[20, 21, 50], but the role of dogs in the transmission of Leishmania species causing cutaneous forms of the disease is not well understood . Observations of long lasting ulcer lesions and a high susceptibility to infection in dogs from Argentina suggested that they could play an important epidemiological role in the transmission of Leishmania parasites species causing cutaneous leishmaniasis . This has important implications for the definition of epidemiological control measures. Indeed, the massive euthanasia of dogs in Brazil has had a questionable efficacy to reduce infection rate in humans , and as a consequence, the World Health Organization has now prioritized the development of veterinary vaccines [34, 41]. Therefore, our model of canine cutaneous leishmaniasis may be very helpful for the evaluation of vaccine candidates. Nonetheless, further development of this animal model may include the evaluation of the addition of sand fly saliva and/or infection by sand fly bites, which have been shown to dramatically alter host immune response to Leishmania infection in mice , even though its effects in dogs have been less straightforward [41, 42].
Analysis of the immune response of L. (L.) mexicana infected dogs indicated an increase in anti-parasite IgG and anti-NH36 IgG levels eight weeks post-infection, which seems much faster than that observed during infection with L. (L.) chagasi/infantum in which case antibody levels took more than a year to increase [42, 50]. However, IgG levels appeared to rise faster when intradermal infection was used . Nonetheless, similar to the visceral form of the disease, for which higher IgG levels are associated with symptomatic disease , we found that a stronger humoral response was associated with larger lesion size. Interestingly, the analysis of IgG isotypes revealed a gradual shift from a predominant IgG2 to a predominant IgG1 isotype over the course of infection, suggesting an initial Th1 type immune response at the beginning on the infection, followed by progression to a Th2 type response at later stages. This change in immune profile would explain the initial control of the lesion leading to its healing, as well as the failure of the immune system to achieve parasite elimination, allowing the detection of Leishmania parasite DNA in the healed lesion and lymph nodes. It is also in agreement with the pathogenic role reported for IgG1 with L. (L.) mexicana infection in murine models . However, cytokine measurements showed a significant IFNγ production in plasma, liver and spleen, while IL-10 was only detected in the liver. A rather mixed Th1/Th2 response is thus more likely to be occurring, as reported in canine visceral leishmaniasis, for which a clear immune polarization has been difficult to observe [43, 55–62]. Nonetheless, a Th1 immune response characterized by predominant IgG2 antibodies and higher IFNγ production seem rather associated with parasite control, as evidenced by a significant association with a reduced splenomegaly, and a tendency to lead to a decreased lesion size and duration. This is in agreement with established mouse models , as well as observations on canine visceral leishmaniasis. The analysis of additional cytokines and tissues, such as draining lymph nodes, should help further characterize the immune response in this animal model.
We present here the first model of canine cutaneous leishmaniasis caused by L. (L.) mexicana. This reproducible model presented typical ulcerated skin lesions in all animals, with characteristics very similar to those observed in naturally infected animals. Parasitological analysis clearly showed the long-term persistence of parasite both in healed skin lesions and the lymph nodes. Also, infection was associated with a mixed Th1/Th2 response, although a Th1 profile seemed to be associated with better parasite control. This model will be very useful to further clarify the role of dogs as reservoirs and in L. (L.) mexicana transmission cycles. Further studies should also provide new insights on the immunopathogenesis of canine cutaneous leishmaniasis. Finally, this model will be very useful for the evaluation of novel vaccine and drug candidates.
This work was funded in part by project PRIORI-2010-004 from the Universidad Autónoma de Yucatán, Yucatan, Mexico. We thank Jorge Canto and Angel Ramos for logistical support and Jesus Valenzuela for critical revisions of the manuscript.
- Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M: Leishmaniasis worldwide and global estimates of its incidence. PloS One. 2012, 7 (5): e35671-PubMed CentralView ArticlePubMedGoogle Scholar
- WHO: Report of the consultative meeting on cutaneous leishmaniasis (WHO/HTM/NTD/IDM/2008.7). 2008, Geneva, Switzerland: WHO, 36-Google Scholar
- WHO: Control of the leishmaniasis: report of a meeting of the WHO committe on the control of the Leishmaniasis. WHO Tech Rep Ser. 2010, 949 (xii-xiii): 1-186.Google Scholar
- Duthie MS, Raman VS, Piazza FM, Reed SG: The development and clinical evaluation of second-generation leishmaniasis vaccines. Vaccine. 2012, 30 (2): 134-141.PubMed CentralView ArticlePubMedGoogle Scholar
- Gonzalez C, Rebollar-Tellez EA, Ibanez-Bernal S, Becker-Fauser I, Martinez-Meyer E, Peterson AT, Sanchez-Cordero V: Current knowledge of Leishmania vectors in Mexico: how geographic distributions of species relate to transmission areas. Am J Trop Med Hyg. 2011, 85 (5): 839-846.PubMed CentralView ArticlePubMedGoogle Scholar
- Bottazzi ME, Dumonteil E, Valenzuela JG, Betancourt-Cravioto M, Tapia-Conyer R, Hotez PJ: Bridging the innovation gap for neglected tropical diseases in Mexico: capacity building for the development of a new generation of antipoverty vaccines. Bol Med Hosp Infant Mex. 2011, 68 (2): 130-138.Google Scholar
- Palatnik-de-Sousa CB: Vaccines for canine leishmaniasis. Front Immunol. 2012, 3: 69-PubMed CentralView ArticlePubMedGoogle Scholar
- Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, Alvar J, Boelaert M: Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?. Nat Rev Microbiol. 2007, 5 (11): 873-882.View ArticlePubMedGoogle Scholar
- Dantas-Torres F: The role of dogs as reservoirs of Leishmania parasites, with emphasis on Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis. Vet Parasitol. 2007, 149 (3–4): 139-146.View ArticlePubMedGoogle Scholar
- Heusser Junior A, Bellato V, Souza AP, Moura AB, Sartor AA, Santos EG, Silva VL: [Canine tegumentar leishmaniasis in the town of Balneario Camboriu in the State of Santa Catarina]. Rev Soc Bras Med Trop. 2010, 43 (6): 713-718.View ArticlePubMedGoogle Scholar
- Dantas-Torres F, de Paiva-Cavalcanti M, Figueredo LA, Melo MF, da Silva FJ, da Silva AL, Almeida EL, Brandao-Filho SP: Cutaneous and visceral leishmaniasis in dogs from a rural community in northeastern Brazil. Vet Parasitol. 2010, 170 (3–4): 313-317.View ArticlePubMedGoogle Scholar
- Miro G, Montoya A, Mateo M, Alonso A, Garcia S, Garcia A, Caballero MJ, Molina R: A leishmaniosis surveillance system among stray dogs in the region of Madrid: ten years of serodiagnosis (1996-2006). Parasitol Res. 2007, 101 (2): 253-257.View ArticlePubMedGoogle Scholar
- Martin-Sanchez J, Morales-Yuste M, Acedo-Sanchez C, Baron S, Diaz V, Morillas-Marquez F: Canine leishmaniasis in southeastern Spain. Emerg Infect Dis. 2009, 15 (5): 795-798.PubMed CentralView ArticlePubMedGoogle Scholar
- Foglia Manzillo V, Di Muccio T, Cappiello S, Scalone A, Paparcone R, Fiorentino E, Gizzarelli M, Gramiccia M, Gradoni L, Oliva G: Prospective study on the incidence and progression of clinical signs in naive dogs naturally infected by Leishmania infantum. PLoS Negl Trop Dis. 2013, 7 (5): e2225-PubMed CentralView ArticlePubMedGoogle Scholar
- Schallig HD, Cardoso L, Semiao-Santos SJ: Seroepidemiology of canine leishmaniosis in Evora (southern Portugal): 20-year trends. Parasit Vectors. 2013, 6: 100-PubMed CentralView ArticlePubMedGoogle Scholar
- Miro G, Cardoso L, Pennisi MG, Oliva G, Baneth G: Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part two. Trends Parasitol. 2008, 24 (8): 371-377.View ArticlePubMedGoogle Scholar
- Costa CH: How effective is dog culling in controlling zoonotic visceral leishmaniasis? A critical evaluation of the science, politics and ethics behind this public health policy. Rev Soc Bras Med Trop. 2011, 44 (2): 232-242.View ArticlePubMedGoogle Scholar
- Aslan H, Dey R, Meneses C, Castrovinci P, Jeronimo SM, Oliva G, Fischer L, Duncan RC, Nakhasi HL, Valenzuela JG, Kamhawi S: A new model of progressive visceral leishmaniasis in hamsters by natural transmission via bites of vector sand flies. J Infect Dis. 2013, 207 (8): 1328-1338.PubMed CentralView ArticlePubMedGoogle Scholar
- Alexander J, Bryson K: T helper (h)1/Th2 and Leishmania: paradox rather than paradigm. Immunol Lett. 2005, 99 (1): 17-23.View ArticlePubMedGoogle Scholar
- Moreno J, Alvar J: Canine leishmaniasis: epidemiological risk and the experimental model. Trends Parasitol. 2002, 18 (9): 399-405.View ArticlePubMedGoogle Scholar
- Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L: Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 2008, 24 (7): 324-330.View ArticlePubMedGoogle Scholar
- Manna L, Reale S, Viola E, Vitale F, Manzillo VF, Michele PL, Caracappa S, Gravino AE: Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol. 2006, 142 (3–4): 271-280.View ArticlePubMedGoogle Scholar
- Carrillo E, Ahmed S, Goldsmith-Pestana K, Nieto J, Osorio Y, Travi B, Moreno J, McMahon-Pratt D: Immunogenicity of the P-8 amastigote antigen in the experimental model of canine visceral leishmaniasis. Vaccine. 2007, 25 (8): 1534-1543.PubMed CentralView ArticlePubMedGoogle Scholar
- Maia C, Campino L: Cytokine and phenotypic cell profiles of Leishmania infantum infection in the dog. J Trop Med. 2012, 2012: 541571-PubMed CentralView ArticlePubMedGoogle Scholar
- Reis AB, Giunchetti RC, Carrillo E, Martins-Filho OA, Moreno J: Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis. Trends Parasitol. 2010, 26 (7): 341-349.View ArticlePubMedGoogle Scholar
- Poot J, Rogers ME, Bates PA, Vermeulen A: Detailed analysis of an experimental challenge model for Leishmania infantum (JPC strain) in dogs. Vet Parasitol. 2005, 130 (1–2): 41-53.View ArticlePubMedGoogle Scholar
- Velez ID, Carrillo LM, Lopez L, Rodriguez E, Robledo SM: An epidemic outbreak of canine cutaneous leishmaniasis in Colombia caused by Leishmania braziliensis and Leishmania panamensis. Am J Trop Med Hyg. 2012, 86 (5): 807-811.PubMed CentralView ArticlePubMedGoogle Scholar
- Santaella J, Ocampo CB, Saravia NG, Mendez F, Gongora R, Gomez MA, Munstermann LE, Quinnell RJ: Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia. Am J Trop Med Hyg. 2011, 84 (5): 674-680.PubMed CentralView ArticlePubMedGoogle Scholar
- Rosete-Ortiz D, Berzunza-Cruz MS, Salaiza-Suazo NL, González C, Treviño-Graza N, Ruiz-Remogio A, Gudiño-Zayas ME, Beltran-Silva S, Romero-Zamora JL, Ugarte-Soto A, Rivas-Sánchez B, Becker I: Canine leishmaniasis in México: the detection of a new focus of canine leishmaniasis in the state of Guerrero correlates with an increase of human cases. Bol Med Hosp Infant Mex. 2011, 68 (2): 88-93.Google Scholar
- Velasco-Castrejón O, Rivas-Sanchez B, Munguia-Saldaña A, Hobart O: Leishmaniasis cutánea de perros en México. Enf Inf Microbiol. 2009, 29 (3): 135-140.Google Scholar
- Figueredo LA, de Paiva-Cavalcanti M, Almeida EL, Brandao-Filho SP, Dantas-Torres F: Clinical and hematological findings in Leishmania braziliensis-infected dogs from Pernambuco, Brazil. Rev Bras Parasitol Vet. 2012, 21 (4): 418-420.View ArticlePubMedGoogle Scholar
- Pirmez C, Coutinho SG, Marzochi MC, Nunes MP, Grimaldi G: Canine American cutaneous leishmaniasis: a clinical and immunological study in dogs naturally infected with Leishmania braziliensis braziliensis in an endemic area of Rio de Janeiro, Brazil. Am J Trop Med Hyg. 1988, 38 (1): 52-58.PubMedGoogle Scholar
- Pirmez C, Marzochi MC, Coutinho SG: Experimental canine mucocutaneous leishmaniasis (Leishmania braziliensis braziliensis). Mem Inst Oswaldo Cruz. 1988, 83 (2): 145-151.View ArticlePubMedGoogle Scholar
- Teixeira MC, Oliveira GG, Santos PO, Bahiense TC, da Silva VM, Rodrigues MS, Larangeira DF, Dos-Santos WL, Pontes-de-Carvalho LC: An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens. Mem Inst Oswaldo Cruz. 2011, 106 (2): 182-189.View ArticlePubMedGoogle Scholar
- Weirather JL, Jeronimo SM, Gautam S, Sundar S, Kang M, Kurtz MA, Haque R, Schriefer A, Talhari S, Carvalho EM, Donelson JE, Wilson ME: Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J Clin Microbiol. 2011, 49 (11): 3892-3904.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang TS, Gawlak SL: Lymphoid organ weights and organ:body weight ratios of growing beagles. Lab Anim. 1989, 23: 143-146.View ArticlePubMedGoogle Scholar
- Cruz-Chan JV, Bolio-González ME, Colin-Flores R, Ramirez-Sierra MJ, Quijano-Hernandez IA, Dumonteil E: Immunopathology of natural Trypanosoma cruzi infection in dogs. Vet Parasitol. 2009, 162: 151-155.View ArticlePubMedGoogle Scholar
- Aguilar-Be I, da Silva Zardo R, Paraguai de Souza E, Borja-Cabrera GP, Rosado-Vallado M, Mut-Martin M, Garcia-Miss MR, de Sousa CB P, Dumonteil E: Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis. Infect Immun. 2005, 73 (2): 812-819.PubMed CentralView ArticlePubMedGoogle Scholar
- Reis AB, Teixeira-Carvalho A, Vale AM, Marques MJ, Giunchetti RC, Mayrink W, Guerra LL, Andrade RA, Correa-Oliveira R, Martins-Filho OA: Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density in Brazilian dogs naturally infected by Leishmania (Leishmania) chagasi. Vet Immunol Immunopathol. 2006, 112 (3–4): 102-116.View ArticlePubMedGoogle Scholar
- Michelin AF, Perri SH, De Lima VM: Evaluation of TNF-alpha, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs. Ann Trop Med Parasitol. 2011, 105 (5): 373-383.View ArticleGoogle Scholar
- Costa DJ, Carvalho RM, Abbehusen M, Teixeira C, Pitombo M, Trigo J, Nascimento F, Amorim L, Abreu-Silva AL, do Socorro Pires Cruz M, Miranda JC, Fukutani K, de Oliveira CI, Barral A, Barral-Netto M, Brodskyn C: Experimental infection of dogs with Leishmania and saliva as a model to study Canine Visceral Leishmaniasis. PloS One. 2013, 8 (4): e60535-PubMed CentralView ArticlePubMedGoogle Scholar
- Paranhos-Silva M, Oliveira GG, Reis EA, de Menezes RM, Fernandes O, Sherlock I, Gomes RB, Pontes-de-Carvalho LC, dos-Santos WL: A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva. Vet Parasitol. 2003, 114 (2): 97-111.View ArticlePubMedGoogle Scholar
- Alves CF, de Amorim IF, Moura EP, Ribeiro RR, Alves CF, Michalick MS, Kalapothakis E, Bruna-Romero O, Tafuri WL, Teixeira MM, Melo MN: Expression of IFN-gamma, TNF-alpha, IL-10 and TGF-beta in lymph nodes associates with parasite load and clinical form of disease in dogs naturally infected with Leishmania (Leishmania) chagasi. Vet Immunol Immunopathol. 2009, 128 (4): 349-358.View ArticlePubMedGoogle Scholar
- Campino L, Santos-Gomes G, Rica Capela MJ, Cortes S, Abranches P: Infectivity of promastigotes and amastigotes of leishmania infantum in a canine model for leishmaniosis [In Process Citation]. Vet Parasitol. 2000, 92 (4): 269-275.View ArticlePubMedGoogle Scholar
- Rallis T, Day MJ, Saridomichelakis MN, Adamama-Moraitou KK, Papazoglou L, Fytianou A, Koutinas AF: Chronic hepatitis associated with canine leishmaniosis (Leishmania infantum): a clinicopathological study of 26 cases. J Comp Pathol. 2005, 132 (2–3): 145-152.View ArticlePubMedGoogle Scholar
- Koutinas AF, Koutinas CK: Pathologic mechanisms underlying the clinical findings in canine leishmaniasis due to Leishmania infantum/chagasi. Vet Pathol. 2014, 51 (2): 527-538.View ArticlePubMedGoogle Scholar
- Aguilar Torrentera F, Lambot MA, Laman JD, Van Meurs M, Kiss R, Noel JC, Carlier Y: Parasitic load and histopathology of cutaneous lesions, lymph node, spleen, and liver from BALB/c and C57BL/6 mice infected with Leishmania mexicana. Am J Trop Med Hyg. 2002, 66 (3): 273-279.PubMedGoogle Scholar
- Ramos-Santos C, Hernandez-Montes O, Sanchez-Tejeda G, Monroy-Ostria A: Visceral leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection. Mem Inst Oswaldo Cruz. 2000, 95 (5): 733-737.View ArticlePubMedGoogle Scholar
- Berzunza-Cruz M, Bricaire G, Romero SZ, Perez-Becker R, Saavedra-Lira E, Perez-Montfort R, Crippa-Rossi M, Velasco-Castrejon O, Becker I: Leishmania mexicana mexicana: genetic heterogeneity of mexican isolates revealed by restriction length polymorphism analysis of kinetoplast DNA. Exp Parasitol. 2000, 95 (4): 277-284.View ArticlePubMedGoogle Scholar
- Maia C, Nunes M, Cristovao J, Campino L: Experimental canine leishmaniasis: clinical, parasitological and serological follow-up. Acta Trop. 2010, 116 (3): 193-199.View ArticlePubMedGoogle Scholar
- Padilla AM, Marco JD, Diosque P, Segura MA, Mora MC, Fernandez MM, Malchiodi EL, Basombrio MA: Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina. Vet Parasitol. 2002, 110 (1–2): 1-10.View ArticlePubMedGoogle Scholar
- Peters NC, Kimblin N, Secundino N, Kamhawi S, Lawyer P, Sacks DL: Vector transmission of leishmania abrogates vaccine-induced protective immunity. PLoS Pathog. 2009, 5 (6): e1000484-PubMed CentralView ArticlePubMedGoogle Scholar
- Travi BL, Osorio EY, Saldarriaga OA, Cadena H, Tabares CJ, Peniche A, Lee S, Melby PC: Clinical, parasitologic, and immunologic evolution in dogs experimentally infected with sand fly-derived Leishmania chagasi promastigotes. Am J Trop Med Hyg. 2009, 81 (6): 994-1003.View ArticlePubMedGoogle Scholar
- Chu N, Thomas BN, Patel SR, Buxbaum LU: IgG1 is pathogenic in Leishmania mexicana infection. J Immunol. 2010, 185 (11): 6939-6946.PubMed CentralView ArticlePubMedGoogle Scholar
- Deplazes P, Smith NC, Arnold P, Lutz H, Eckert J: Specific IgG1 and IgG2 antibody responses of dogs to Leishmania infantum and other parasites. Parasite Immunol. 1995, 17 (9): 451-458.View ArticlePubMedGoogle Scholar
- Iniesta L, Gallego M, Portus M: Immunoglobulin G and E responses in various stages of canine leishmaniosis. Vet Immunol Immunopathol. 2005, 103 (1–2): 77-81.View ArticlePubMedGoogle Scholar
- Reis AB, Martins-Filho OA, Teixeira-Carvalho A, Carvalho MG, Mayrink W, Franca-Silva JC, Giunchetti RC, Genaro O, Correa-Oliveira R: Parasite density and impaired biochemical/hematological status are associated with severe clinical aspects of canine visceral leishmaniasis. Res Vet Sci. 2006, 81 (1): 68-75.View ArticlePubMedGoogle Scholar
- de Almeida MC, Vilhena V, Barral A, Barral-Netto M: Leishmanial infection: analysis of its first steps: a review. Mem Inst Oswaldo Cruz. 2003, 98 (7): 861-870.View ArticlePubMedGoogle Scholar
- Teixeira Neto RG, Giunchetti RC, Carneiro CM, Vitor RW, Coura-Vital W, Quaresma PF, Ker HG, de Melo LA, Gontijo CM, Reis AB: Relationship of Leishmania-specific IgG levels and IgG avidity with parasite density and clinical signs in canine leishmaniasis. Vet Parasitol. 2010, 169 (3–4): 248-257.View ArticlePubMedGoogle Scholar
- Boggiatto PM, Ramer-Tait AE, Metz K, Kramer EE, Gibson-Corley K, Mullin K, Hostetter JM, Gallup JM, Jones DE, Petersen CA: Immunologic indicators of clinical progression during canine Leishmania infantum infection. Clin Vaccine Immunol: CVI. 2010, 17 (2): 267-273.PubMed CentralView ArticlePubMedGoogle Scholar
- Menezes-Souza D, Correa-Oliveira R, Guerra-Sa R, Giunchetti RC, Teixeira-Carvalho A, Martins-Filho OA, Oliveira GC, Reis AB: Cytokine and transcription factor profiles in the skin of dogs naturally infected by Leishmania (Leishmania) chagasi presenting distinct cutaneous parasite density and clinical status. Vet Parasitol. 2011, 177 (1–2): 39-49.View ArticlePubMedGoogle Scholar
- Cardoso L, Schallig HD, Cordeiro-da-Silva A, Cabral M, Alunda JM, Rodrigues M: Anti-Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs. Vet Immunol Immunopathol. 2007, 117 (1–2): 35-41.View ArticlePubMedGoogle Scholar
- Mansueto P, Vitale G, Di Lorenzo G, Rini GB, Mansueto S, Cillari E: Immunopathology of leishmaniasis: an update. Int J Immunopathol Pharmacol. 2007, 20 (3): 435-445.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.