Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the L. mexicana complex (L. amazonensis, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis/L. peruviana possible hybrids, two L. amazonensis corresponded to L. mexicana, and three L. garnhami and two L. amazonensis corresponded to L. mexicana/L. amazonensis possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area.

Genetic profiling of HSP70 gene in local Iraqi goats.

Animals display numerous physiological and behavioral responses that reduce the effects of heat stress. Moreover, genetic variance is strongly associated with responses to heat stress, including variants of heat shock proteins (HSPs) that are necessary for thermoregulation and stress resistance. Herein, we performed the molecular profiling of the HSP70 gene, and its polymorphism was demonstrated as a possible factor in the stress tolerance of local Iraqi goats. A number of different mutations were found owing to seven main polymorphisms. Results indicated the occurrence of silent and missense mutations in sequences obtained for Iraqi local goats. Genetic diversity was observed in the HSP70 gene of Iraqi local goats on the basis of phylogenetic-tree analysis as some mutations occurred once whereas others occurred multiple times. The polymorphisms LC616787, LC616788, and LC616791 were combined with the reference gene in the same branch, whereas polymorphisms (LC616785 and LC616786) and (LC616789 and LC616790) met in different branches, respectively. Moreover, all studied proteins had mismatches in their three-dimensional structures. Therefore, the presence of specific genetic differences within the HSP70 gene in Iraqi goats can increase the possibility of selecting animals more suitable to various levels of stress.

Os animais apresentam uma série de respostas fisiológicas e comportamentais que reduzem os efeitos do estresse térmico. Além disso, a variância genética está fortemente associada às respostas ao estresse térmico, incluindo variantes de proteínas de choque térmico (HSPs) que também são necessárias para a termorregulação e resistência ao estresse. O perfil molecular do gene HSP70 foi realizado neste estudo e o polimorfismo desse gene foi demonstrado como um possível fator na tolerância ao estresse de caprinos iraquianos. Várias mutações diferentes foram encontradas devido a sete polimorfismos principais. Os resultados indicam a ocorrência de mutações silenciosas e sem sentido em sequências obtidas para caprinos iraquianos. A diversidade genética pode ser vista no gene HSP70 de cabras locais iraquianas com base na análise da árvore filogenética, já que algumas mutações ocorreram uma vez, enquanto outras ocorreram várias vezes. Os polimorfismos LC616787, LC616788 e LC616791 foram combinados com o gene de referência no mesmo ramo, enquanto os polimorfismos (LC616785 e LC616786) e (LC616789 e LC616790) se encontraram em diferentes ramos, respectivamente. O estudo também revelou que todas as proteínas estudadas tinham incompatibilidade sem suas estruturas tridimensionais. De acordo com nossas descobertas, a presença de diferenças genéticas específicas dentro do gene HSP70 em caprinos iraquianas aumentaria a possibilidade de seleção de animais mais adequados a vários níveis de estresse.

Tegumentary leishmaniasis by Leishmania braziliensis complex in Cochabamba, Bolivia including the presence of L. braziliensis outlier: Tegumentary leishmaniasis in Cochabamba, Bolivia: Tegumentary leishmaniasis in Cochabamba, Bolivia.

Leishmaniasis is caused by protozoans of the Leishmania genus, which includes more than 20 species capable of infecting humans worldwide. In the Americas, the most widespread specie is L. braziliensis, present in 18 countries including Bolivia. The taxonomic position of the L. braziliensis complex has been a subject of controversy, complicated further by the recent identification of a particular subpopulation named L. braziliensis atypical or outlier. The aim of this study was to carry out a systematic analysis of the L. braziliensis complex in Bolivia and to describe the associated clinical characteristics. Forty-one strains were analyzed by sequencing an amplified 1245 bp fragment of the hsp70 gene, which allowed its identification as: 24 (59%) L. braziliensis, 16 (39%) L. braziliensis outlier, and one (2%) L. peruviana. In a dendrogram constructed, L. braziliensis and L. peruviana are grouped in the same cluster, whilst L. braziliensis outlier appears in a separate branch. Sequence alignment allowed the identification of five non-polymorphic nucleotide positions (288, 297, 642, 993, and 1213) that discriminate L. braziliensis and L. peruviana from L. braziliensis outlier. Moreover, nucleotide positions 51 and 561 enable L. peruviana to be discriminated from the other two taxa. A greater diversity was observed in L. braziliensis outlier than in L. braziliensis-L. peruviana. The 41 strains came from 32 patients with tegumentary leishmaniasis, among which 22 patients (69%) presented cutaneous lesions (11 caused by L. braziliensis and 11 by L. braziliensis outlier) and 10 patients (31%) mucocutaneous lesions (eight caused by L. braziliensis, one by L. braziliensis outlier, and one by L. peruviana). Nine patients (28%) simultaneously provided two isolates, each from a separate lesion, and in each case the same genotype was identified in both. Treatment failure was observed in six patients infected with L. braziliensis and one patient with L. peruviana.

Arthrospira (Spirulina) Platensis Can Be Considered as a Probiotic Alternative to Reduce Heat Stress in Laying Japanese Quails

This study was conducted to investigate the effects of Arthrospira platensis (Spirulina platensis, SP) on feed intake, feed conversion ratio, egg weight, hen day egg production, intestinal microflora, heat stress biomarkers, and HSP70 gene expression in laying Japanese quails (Coturnix coturnix japonica) suffering heat stress condition. A total of 250 female quails were allocated to 5 treatments, 5 replicates and 10 birds in each replicate in a completely randomized design. Experimental treatments included: 1) basal diet, 2) basal diet+ 0.03 % probiotic, 3) basal diet+ 0.1 % SP, 4) basal diet+ 0.3 % SP, 5) basal diet+ 0.5 % SP. During the last 6 days of the experiment, the quails were exposed to 8h of 34±1°C. The birds had free access to feed and water during the experiment. The results showed that using probiotic and different levels of Spirulina had no significant effect on laying performance of Japanese quails (p>0.05). Probiotic supplement increased Lactobacil bacteria population in laying quails ileum under heat stress (p 0.05). Different levels of SP decreased Escherichia coli population in laying quails ileum suffering heat stress (p 0.05).SP at the level of 0.5% caused the lowest blood Malondialdehyde level, heterophil, and H/L ratio (p 0.05).However, HSP70 gene expression in the heart or the liver of laying quails was not different (p>0.05). In conclusion, the results of the present study revealed that SP at the level of 0.5 % has the potential to be considered as a probiotic alternative in the diet of laying quails suffering heat stress condition.

Arthrospira (Spirulina) Platensis Can Be Considered as a Probiotic Alternative to Reduce Heat Stress in Laying Japanese Quails

This study was conducted to investigate the effects of Arthrospira platensis (Spirulina platensis, SP) on feed intake, feed conversion ratio, egg weight, hen day egg production, intestinal microflora, heat stress biomarkers, and HSP70 gene expression in laying Japanese quails (Coturnix coturnix japonica) suffering heat stress condition. A total of 250 female quails were allocated to 5 treatments, 5 replicates and 10 birds in each replicate in a completely randomized design. Experimental treatments included: 1) basal diet, 2) basal diet+ 0.03 % probiotic, 3) basal diet+ 0.1 % SP, 4) basal diet+ 0.3 % SP, 5) basal diet+ 0.5 % SP. During the last 6 days of the experiment, the quails were exposed to 8h of 34±1°C. The birds had free access to feed and water during the experiment. The results showed that using probiotic and different levels of Spirulina had no significant effect on laying performance of Japanese quails (p>0.05). Probiotic supplement increased Lactobacil bacteria population in laying quails ileum under heat stress (p 0.05). Different levels of SP decreased Escherichia coli population in laying quails ileum suffering heat stress (p 0.05).SP at the level of 0.5% caused the lowest blood Malondialdehyde level, heterophil, and H/L ratio (p 0.05).However, HSP70 gene expression in the heart or the liver of laying quails was not different (p>0.05). In conclusion, the results of the present study revealed that SP at the level of 0.5 % has the potential to be considered as a probiotic alternative in the diet of laying quails suffering heat stress condition.(AU)

Development of a real-time PCR assay for detection of Cryptosporidium canis in dog fecal samples.

Cryptosporidiosis is an emerging zoonotic disease caused by the worldwide distributed parasitic protozoa Cryptosporidium spp. The host-adapted species Cryptosporidium canis is most frequently found in dogs, although human infections with this species have been described. This study aimed to develop a real-time PCR targeting the HSP70 protein gene for C. canis DNA detection in dog fecal samples collected from two municipalities in the state of São Paulo, Brazil. Furthermore, the occurrence of Cryptosporidium spp. and. C. canis was also determined by nested PCR. Fecal samples from 367 dogs (21 puppies and 346 adults) were purified by water-ether sedimentation. A real-time PCR protocol targeting the HSP70 gene for the species-specific detection of C. canis was developed and compared with nested PCR results. Real-time PCR identified C. canis in 15.3% (58/367) samples. Nested PCR revealed that 10.4% (38/367) of samples were positive for Cryptosporidium spp. All sequenced 18S rRNA amplicons were C. canis. There was a higher prevalence of Cryptosporidium spp. and C. canis in puppies compared to adult dogs. No non-specific amplification was observed in C. canis specific real-time PCR assay.

Distinct genetic profiles of Leishmania (Viannia) braziliensis associate with clinical variations in cutaneous-leishmaniasis patients from an endemic area in Brazil.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.