Jeffrey J. Shaw

Infectiousness in a Cohort of Brazilian Dogs: Why Culling Fails to Control Visceral Leishmaniasis in Areas of High Transmission

The elimination of seropositive dogs in Brazil has been used to control zoonotic visceral leishmaniasis but with little success. To elucidate the reasons for this, the infectiousness of 50 sentinel dogs exposed to natural Leishmania chagasi infection was assessed through time by xenodiagnosis with the sandfly vector, Lutzomyia longipalpis. Eighteen (43%) of 42 infected dogs became infectious after a median of 333 days in the field (105 days after seroconversion). Seven highly infectious dogs (17%) accounted for >80% of sandfly infections. There were positive correlations between infectiousness and anti-Leishmania immunoglobulin G, parasite detection by polymerase chain reaction, and clinical disease (logistic regression, r2=0.08-0.18). The sensitivity of enzyme-linked immunosorbent assay to detect currently infectious dogs was high (96%) but lower in the latent period (<63%), and specificity was low (24%). Mathematical modeling suggests that culling programs fail because of high incidence of infection and infectiousness, the insensitivity of the diagnostic test to detect infectious dogs, and time delays between diagnosis and culling.

Wild and synanthropic hosts of Leishmania (Viannia) braziliensis in the endemic cutaneous leishmaniasis locality of Amaraji, Pernambuco State, Brazil

Evidence of Leishmania infection was found in small mammals captured between 1996 and 2000 in the Amaraji region, Pernambuco State, Brazil. The kDNA polymerase chain reaction (PCR), using primers specific for subgenus L. (Viannia), was positive for 43/153 water rats (Nectomys squamipes), 13/81 black rats (Rattus rattus), 15/103 grass mice (Bolomys lasiurus), 1/14 marsh mice (Holochilus scieurus), 2/50 field mice (Akodon arviculoides), 2/12 woolly opossums (Marmosa sp.), and 5/37 common opossums (Didelphis albiventris). This same kDNA PCR was positive for 12/61 dog and 8/58 horse skin samples. In paired PCR tests of 203 small mammals, 18.7% were positive with the kDNA primers and 18.2% with rDNA primers. Amastigotes were seen in 26/460 and L. (V.) braziliensis was isolated from 5 grass mice and 1 black rat. We concluded that small mammals, particularly rodents, are infected with parasites of the subgenus L. (Viannia). The isolation of L. (V.) braziliensis zymodeme IOC/Z74 from 6 rodents and the fact that all the other described L. (Viannia) species that commonly infect humans have never been found in rodents or marsupials leads us to suggest that the positive PCRs indicate infections of L. (V.) braziliensis. The isolation of zymodeme IOC/Z74 from humans reinforces our hypothesis that small, ground-loving mammals, such as rodents are the primary reservoirs of L. (V.) braziliensis.

Evidence for a cluster of genes on chromosome 17q11–q21 controlling susceptibility to tuberculosis and leprosy in Brazilians

The region of conserved synteny on mouse chromosome 11/human 17q11–q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Zlr score 2.34; P=0.01) and D17S1795 (Zlr 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Zlr 2.04; P=0.02). Combined analysis shows significant linkage (peak Zlr 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A–8.4 Mb–CCL18–32.3 kb–CCL4–6.04 Mb–STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

The dermal leishmaniases of Brazil, with special reference to the eco-epidemiology of the disease in Amazonia

Six species of Leishmania are at present known to cause cutaneous and/or mucocutaneous leishmaniasis in Brazil, and they are all to be found in the Amazon region of this country. The eco-epidemiology of each is discussed, with the observation that the Amazonian leishmaniases are all zoonoses, with their source in silvatic mammals and phlebotomine sandfly vectors. With mans destruction of the natural forest in southern Brazil, some sandfly species have survived by adapting to a peridomestic or domiciliary habitat in rural areas. Some domestic animals, such as dogs and equines are seemingly now involved in the epidemiology of the disease. No such process has yet been reported in the Amazon region, but may well take place with the continuing devastation of its forest.

Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs.

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78-88%) 0-135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93-100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 × 10−5; D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

Taxonomy of the genus Leishmania: present and future trends and their implications

The application of different taxonomic methods (Cladistic, Evolutionary Taxonomy and Numerical Taxonomy) to the taxonomy of the Genus Leishmania are reviewed. The major groupings of the most recent classifications obtained using the cladistical approach agree with the major divisions of previous classifications which used traditional taxonomy (Evolutionary Taxonomy). The advantage of the cladistical approach is that it produces cladograms whose branches indicate more accurately levels of relationships between the different taxa. Numerical Taxonomy is useful for identification but not as good as the cladistical approach for classification. The ancient division of this monophyletic genus into two major evolutionary lines supports the use of the subgeneric names Leishmania and Viannia.

New PCR Assay Using Glucose-6-Phosphate Dehydrogenase for Identification of Leishmania Species

ABSTRACT Glucose-6-phosphate dehydrogenase (G6PD) is one of the multilocus enzymes used to identify Leishmania by zymodeme analysis. The polymorphic pattern revealed by partial characterization of the gene encoding G6PD generated molecular markers useful in the identification of different Leishmania species by PCR. Initially degenerate oligonucleotides were designed on the basis of data on the conserved active center described for other organisms. Primers for reverse transcription-PCR experiments, designed from the nucleotide sequence of the PCR product, enabled us to characterize the 5′ and 3′ untranslated regions and the G6PD open reading frame of reference strains of Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) amazonensis. Sets of paired primers were designed and used in PCR assays to discriminate between the parasites responsible for tegumentar leishmaniasis of the subgenera Leishmania (Leishmania) and Leishmania (Viannia) and to distinguish L. (Viannia) braziliensis from others organisms of the subgenus Leishmania (Viannia). No amplification products were detected for the DNA of Crithidia fasciculata, Trypanosoma cruzi, or Leishmania (Sauroleishmania) tarentolae or DNA from a healthy human control. The tests proved to be specific and were sensitive enough to detect parasites in human biopsy specimens. The successful discrimination of L. (Viannia) braziliensis from other parasites of the subgenus Leishmania (Viannia) opens the way to epidemiological studies in areas where more than one species of the subgenus Leishmania (Viannia) exist, such as Amazonia, as well as follow-up studies after chemotherapy and assessment of clinical prognoses.

Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi

30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.

Host preferences of the phlebotomine sandfly Lutzomyia longipalpis in Amazonian Brazil

Abstract. Experiments were undertaken to determine the relative attractiveness of humans, dogs and chickens to Lutzomyia longipalpis, the principal vector of Leishmania chagasi causing American visceral leishmaniasis Field experiments in two villages on Marajo Island, Para State, Brazil, showed that one boy attracted significantly more flies than one dog or chicken, and slightly fewer flies than a group of six chickens. Experiments with laboratory‐bred female flies showed that a significantly greater number of flies engorged on a single human than on either a single dog or chicken, and man‐biting catches demonstrated the willingness of flies to bite in the field. It appears that Lu. longipalpis has catholic feeding habits, the attractiveness of different hosts being largely a function of their relative sizes. These results are discussed with reference to the epidemiology of visceral leishmaniasis in Brazil.