Isolation and molecular identification of Leptospira santarosai and Leptospira interrogans in equines from eastern Mexico.

Leptospirosis is an infectious disease with a worldwide distribution, which represents a major challenge in animal production across developing countries, mainly in tropical areas. Horses are particularly susceptible to the disease, presenting manifestations ranging from subclinical to the development of uveitis that compromises the visual health of the animals. In recent years, serological studies have been carried out in equid populations from America, demonstrating high exposure. For this reason, the aim of this study was to demonstrate microbiologically and molecularly the presence of the members of the genus Leptospira in urine samples from equids in an endemic state of leptospirosis in Mexico, and to detect the serological presence of anti-Leptospira antibodies in the sampled animals. For this reason, blood and urine samples were collected from 28 horses and one mule from three localities in the state of Veracruz, Mexico. Urine samples were inoculated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and the recovered isolates were typed using a short Multi Locus Sequence Typing scheme. Amplifications of the expected size were subjected to sequencing, and the recovered sequences were compared with those of reference deposited in GenBank using the BLAST tool. To identify their phylogenetic position, we performed a phylogenetic reconstruction using the maximum likelihood method. Additionally, Microscopic Agglutination test was performed on the serum samples to identify anti-Leptospira antibodies. We recovered 16 urine isolates which tested positive for the presence of Leptospira DNA. The phylogenetic reconstruction and the MLST analysis confirmed the presence of several genotypes of Leptospira interrogans and Leptospira santarosai. An overall serological frequency of 97.1% was detected. Our results represent the first record of the presence of Leptospira through bacteriological isolates in equids from Mexico.

First confirmed case of equine pythiosis in Northern Veracruz, Mexico.

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico.

Unraveling the diversity of Trypanosoma species from Central Mexico: Molecular confirmation on the presence of Trypanosoma dionisii and novel Neobat linages.

Bats are one of the groups of mammals with the highest number of associated Trypanosoma taxa. There are 50 Trypanosoma species and genotypes infecting more than 75 species of bats across five continents. However, in Mexico, the inventory of species of the genus Trypanosoma associated with bats is limited to only two species (Trypanosoma vespertilionis and Trypanosoma cruzi) even though 140 species of bats inhabit this country. Specifically, 91 bat species have been recorded in the state of Veracruz, but records of trypanosomatids associated with this mammalian group are absent. Due to the complex Trypanosoma-bat relationship, the high diversity of bat species in Veracruz, as well as the lack of records of trypanosomatids associated with bats for this state, the aim of this work was to analyze the diversity of species of the genus Trypanosoma and their presence from a bat community in the central area of the state of Veracruz, Mexico. During the period of January to August 2022 in the Tequecholapa Environmental Management Unit where bats were collected using mist nets and blood samples were obtained from their thumbs. We extracted genetic material and amplified a fragment of 800 bp of the 18S ribosomal gene of the genus Trypanosoma by conventional PCR. The positive amplicons were sequenced, and phylogenetic reconstruction was performed to identify the parasite species. A total of 285 bats (149♀, 136♂) belonging to 13 species from 10 genera and a single family (Phyllostomidae) were collected. Twenty-three specimens from six species tested positive for the presence of Trypanosoma dionisii, Trypanosoma sp. Neobat 4, and a potential novelty species provisionally named as Trypanosoma sp. Neobat 6. The results of the present work increase the number of species of the genus Trypanosoma infecting bats in Mexico and in the Neotropical region.

Entomological Survey and Leishmania (Leishmania) mexicana Prevalence in Sand Fly Species during an Outbreak of Cutaneous Leishmaniasis in Quintana Roo State, Mexico.

(1) Background: Localized cutaneous leishmaniasis is a neglected vector-borne disease that has become a serious public health problem in the Yucatan Peninsula. Although more than 60% of cases originate from the state of Quintana Roo, it is one of the least explored areas in terms of incriminating vectors of the Leishmania parasite. Additionally, cases of leishmaniasis have increased substantially in that region in recent years. For this reason, we explored and provided primary evidence of Leishmania DNA in sand fly species from four localities during outbreaks of leishmaniasis in Quintana Roo. We also contributed information on the regional genetic diversity of Leishmania parasites. (2) Methods: Sand flies were collected during several periods from November 2022 to April 2023 using Mosquito Light Circle and Shannon traps, as well as an active entomological search in refuges. For Leishmania detection, we amplified a fragment of 300-350 bp of the internal transcribed spacer subunit 1 (ITS-1). (3) Results: Of the 242 females collected, we detected Leishmania DNA in 25 specimens represented by Bichromomyia olmeca (1), Psathyromyia shannoni (17), Lutzomyia cruciata (4), Psathyromyia undulata (2), and Dampfomyia deleoni (1). The detection of Leishmania in these last two species represents new records for the Yucatan Peninsula and for Mexico. Leishmania (Leishmania) mexicana was the only species detected in the Phlebotominae species, with prevalence values that ranked between 7.41% and 33.33% from specimens collected in the sylvatic areas of Cozumel Island and Petcacab. (4) Conclusions: This study provides the first evidence of infection of Da. deleoni and Pa. undulata by L. (L.) Mexicana. In addition, the presence of three dominant haplotypes in all the evaluated localities was evidenced using the analysis of genetic diversity, and the locality of Petcacab was the one with the circulation of two new haplotypes not previously described in Mexico or neighboring countries. These results highlight the importance of intensive epidemiological surveillance due to the dynamics of transmission of Leishmania between different species.

Molecular detection of hemotropic Mycoplasma and Bartonella species in lice from sheep and goats of Mexico.

The knowledge of lice associated with small ruminants, especially sheep and goats, is scarce. In Mexico, there are historical reports of six species of chewing and sucking lice associated with Capra hircus and Ovis canadensis. However, the reports did not analyze the ecology of the infestations or the presence of potentially pathogenic bacteria. For this reason, the objectives of this study were i) to identify the species of lice associated with sheep and goats in three states of the Mexican Republic, ii) to characterize the infestations, and iii) to identify the presence of bacterial pathogens. From October 2019 to August 2021, six ranches with sheep and goats were sampled in the states of Hidalgo and Veracruz. Hosts were visually inspected, and lice were retrieved with forceps. The specimens were sexed and identified using morphological taxonomic keys. DNA extraction was performed individually, and a fragment of the cytochrome oxidase subunit 1 gene (COI) was amplified for the molecular identification of the specimens. Subsequently, Anaplasma, Bartonella, Ehrlichia, Mycoplasma, and Rickettsia were molecularly detected. Additionally, the infestations were characterized by calculating the prevalence and mean abundances. We collected 563 specimens of three species, Bovicola caprae, Bovicola ovis, and Linognathus africanus. The highest infestation levels were recorded for B. ovis (66.7%; 4.4) from Veracruz. Additionally, two Bartonella species were detected: Bartonella mellophagi in B. ovis and Bartonella capreoli in L. africanus. In contrast, Mycoplasma ovis was detected exclusively in one pool of B. ovis. This study provides new bacterial-ectoparasite associations and highlights the possible role of these neglected ectoparasites as vectors in the populations of sheep and goats from Mexico.

DNA barcoding and new records of Ornithodoros yumatensis from Central Mexico.

Bats represent the second order of mammals with the highest number of species worldwide with over 1,616 species, and almost 10% of them are recorded in Mexico. These mammals have a great diversity of ectoparasites, in particular soft ticks of the genus Ornithodoros. Desmodus rotundus is one of the bat species that has scarcely been studied in terms of tick species richness in Mexico, with three tick species reported in five of the 32 Mexican states. For this reason, the aim of the present work was to identify ticks associated with D. rotundus from Central Mexico. Fieldwork was undertaken in the municipality El Marqués, Ejido Atongo A, Querétaro, Mexico. Bats were captured using mist nets and were visually inspected for tick presence. The ectoparasites were identified morphologically and molecularly with the use of mitochondrial markers 16SrDNA and cytochrome oxidase subunit I (COI). A total of 30 D. rotundus (1 female, 29 males) were captured, from which 20 larvae identified as Ornithodoros yumatensis were recovered. Molecular analysis confirmed the presence of this species with identity values of 99-100% with sequences of this species from the southwestern US, and the Yucatán Peninsula, Mexico. This is the first report of ticks associated with bats for the state of Querétaro, providing the first sequences of the COI gene from Mexican populations of O. yumatensis and shows an increase in the distribution of this soft tick across Central Mexico.

Novel Francisella-like endosymbiont and Anaplasma species from Amblyomma nodosum hosted by the anteater Tamandua Mexicana in Mexico.

The microbiome represents a complex network among the various members of the community of microorganisms that are associated with a host. The composition of the bacterial community is essential to supplement multiple metabolic pathways that the host lacks, particularly in organisms with blood-sucking habits such as ticks. On the other hand, some endosymbionts showed some competence with potentially pathogenic microorganisms. Francisella-like endosymbionts (FLEs) encompass a group of gamma-proteobacterias that are closely related to Francisella tularensis, but are usually apathogenic, which brings nutrients like vitamin B and other cofactors to the tick. It has been postulated that the main route of transmission of FLE is vertical; however, evidence has accumulated regarding the possible mechanism of horizontal transmission. Despite growing interest in knowledge of endosymbionts in the Neotropical region, the efforts related to the establishment of their inventory for tick communities are concentrated in South and Central America, with an important gap in knowledge in Mesoamerican countries such as Mexico. For this reason, the aim of this work was to evaluate the presence and diversity of endosymbionts in the highly host-specialized tick Amblyomma nodosum collected from the anteater Tamandua mexicana in Mexico. We analysed 36 A. nodosum for the presence of DNA of endosymbiont (Coxiella and Francisella) and pathogenic (Anaplasma, Borrelia, Ehrlichia and Rickettsia) bacteria. The presence of a member of the genus Francisella and Candidatus Anaplasma brasiliensis was demonstrated. Our findings provide information on the composition of A. nodosum's microbiome, increasing the inventory of bacterial species associated with this hard tick on the American continent.

A story of a lone star tick: an imported case of Amblyomma americanum (Linnaeus, 1758) infected with Rickettsia amblyommatis that parasitized a US traveler returning to Mexico.

In this study, we report the presence of a female Amblyomma americanum tick attached to a former resident of the East Coast of the United States who moved to Mexico city. The amplification and sequencing of gene fragments of the 16S-rDNA and cytochrome c oxidase subunit 1 corroborated the identification of the species of the tick. Additionally, the presence of DNA of Rickettsia amblyommatis was confirmed. This work is the first report of an exotic tick of the genus Amblyomma in a traveler from the US to Mexico and represents the second record of an imported tick attached to humans in Mexico.

Identification of the G184C, C190A and T2134A mutations in the para-sodium channel gene of the southern cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to cypermethrin in northern Veracruz, Mexico.

The southern cattle tick (Rhipicephalus microplus) represents one of the ectoparasites with the greatest distribution worldwide. Infestations by this arthropod can cause a decrease in the production of meat and milk, as well as anaemia and the transmission of bacterial and parasitic agents. For this reason, several active molecules have been developed to control these arthropods. A widely used group of ixodicides are pyrethroids, especially cypermethrin, which have knockdown effects on ticks. Resistance to cypermethrin has been reported in ticks since the 2000s; it was registered for the first time in Mexico in 2009. Even though multiple studies have evaluated resistance with conventional tests, there are few studies in Mexico that have identified the presence of single nucleotide polymorphisms (SNPs) associated with resistance. Hence, the aim of this work was to monitor three mutations associated with resistance in the sodium/chlorine channel in eight populations of ticks from northern Veracruz. Engorged adult females were collected from which genomic DNA was extracted. Subsequently, three mutations in domains II and III of para­sodium channel gene were detected by conventional PCR and sequencing. Global alignments were done with the reference sequences deposited in GenBank. A total of 116 engorged females were analysed, of which 10 tested positive for G184C and C190A of domain II of the para­sodium channel gene. T2134A was present in domain III in a single production unit. This is the first work where molecular monitoring of cypermethrin resistance has been carried out in the northern zone of the state of Veracruz.

The Low Variability of Tc24 in Trypanosoma cruzi TcI as an Advantage for Chagas Disease Prophylaxis and Diagnosis in Mexico.

(1) Background: Chagas disease is the main neglected tropical disease in America. It is estimated that around 6 million people are currently infected with the parasite in Latin America, and 25 million live in endemic areas with active transmission. The disease causes an estimated economic loss of USD 24 billion dollars annually, with a loss of 75,200 working years per year of life; it is responsible for around ~12,000 deaths annually. Although Mexico is an endemic country that recorded 10,186 new cases of Chagas disease during the period of 1990-2017, few studies have evaluated the genetic diversity of genes that could be involved in the prophylaxis and/or diagnosis of the parasite. One of the possible candidates proposed as a vaccine target is the 24 kDa trypomastigote excretory-secretory protein, Tc24, whose protection is linked to the stimulation of T. cruzi-specific CD8+ immune responses. (2) Methods: The aim of the present study was to evaluate the fine-scale genetic diversity and structure of Tc24 in T. cruzi isolates from Mexico, and to compare them with other populations reported in the Americas with the aim to reconsider the potential role of Tc24 as a key candidate for the prophylaxis and improvement of the diagnosis of Chagas disease in Mexico. (3) Results: Of the 25 Mexican isolates analysed, 48% (12) were recovered from humans and 24% (6) recovered from Triatoma barberi and Triatoma dimidiata. Phylogenetic inferences revealed a polytomy in the T. cruzi clade with two defined subgroups, one formed by all sequences of the DTU I and the other formed by DTU II-VI; both subgroups had high branch support. Genetic population analysis detected a single (monomorphic) haplotype of TcI throughout the entire distribution across both Mexico and South America. This information was supported by Nei's pairwise distances, where the sequences of TcI showed no genetic differences. (4) Conclusions: Given that both previous studies and the findings of the present work confirmed that TcI is the only genotype detected from human isolates obtained from various states of Mexico, and that there is no significant genetic variability in any of them, it is possible to propose the development of in silico strategies for the production of antigens that optimise the diagnosis of Chagas disease, such as quantitative ELISA methods that use this region of Tc24.

Molecular detection of Rickettsia felis in fleas and ticks collected from dogs and cats of Puebla, Mexico.

Fleas and ticks represent the two main groups of ectoparasites that infest companion animals. In particular, the flea Ctenocephalides felis felis and several members of the Rhipicephalus sanguineus complex are the main vectors of a wide range of pathogens on the American continent. They are competent vectors for several members of the genus Rickettsia, which encompass at least 15 pathogenic obligate intracellular bacteria that colonize the endothelial cells of vertebrates. In Mexico, 10 species of Rickettsia belonging to three groups have been detected in six species of ectoparasites from dogs in 9 of the 32 states of the country. However, in some larger regions of the country, active epidemiological surveillance has not been carried out. For this reason, the aim of this study was to identify the presence of members of the genus Rickettsia in fleas and ticks of dogs and cats in the state of Puebla, Mexico. A cross-sectional study was carried out to collect ectoparasites of dogs and cats during August to November 2019. Samples were fixed in 70% ethanol and examined to identify the presence of Rickettsia DNA by the amplification and sequencing of specific fragments of the gltA and ompB genes using conventional PCR. The recovered sequences were compared with those deposited in GenBank, and phylogenetic analyses were carried out to identify the position of the pathogens detected with respect to the valid species previously reported worldwide. Additionally, ecological parameters of the ectoparasite infestations were also calculated. We recovered 196 ectoparasites belonging to two species, 33 C. felis felis and 163 R. sanguineus s.l. (Rhipicephalus linnaei), parasitizing 46 hosts (42 dogs and 4 cats) in 11 localities of the state of Puebla. We detected the presence of Rickettsia felis in three pools of C. felis felis, and five from R. sanguineus s.l. Our work provides the first record of R. felis in hard ticks of Mexico and Central America, with new collection localities for this pathogen in central Mexico.

Confirmation of the presence of Rickettsia felis infecting Ornithodoros puertoricensis in Mexico.

Soft ticks are neglected competent vectors of a wide range of pathogenic microorganisms, among which bacteria of the genera Rickettsia and Borrelia stand out. In Mexico, previous studies have shown the presence of a member of the Ornithodoros talaje complex in the Virginia opossum (Didelphis virginiana Didelphimorphia: Didelphidae Kerr) from southeastern Mexico. However, its specific identification has not been achieved. Two D. virginiana were treated in a private clinic during the period of April-May 2022. Tick larvae were manually removed, DNA extraction was performed, and some genes from various bacterial and parasitic pathogens were amplified and sequenced. A total of 96 larvae were recovered, which were morphologically identified as Ornithodoros puertoricensis (Ixodida: Argasidae Fox); the 16 S sequences showed a similarity of 96.79%-99.51% with sequences of O. puertoricensis from Panama and Colombia. The presence of Rickettsia felis (Rickettsiales: Rickettsiaceae Bouyer et al.) was detected in 15 specimens from one host. The soft tick O. puertoricensis is recorded for the first time as an ectoparasite of the Virginia opossum in America and represents the second report for this soft tick in Mexico since 1963. This represents the most northern record of this tick species in its geographic distribution and brings a new soft tick-Rickettsia association.

A story of a lone star tick: an imported case of Amblyomma americanum (Linnaeus, 1758) infected with Rickettsia amblyommatis that parasitized a US traveler returning to Mexico

ABSTRACT In this study, we report the presence of a female Amblyomma americanum tick attached to a former resident of the East Coast of the United States who moved to Mexico city. The amplification and sequencing of gene fragments of the 16S-rDNA and cytochrome c oxidase subunit 1 corroborated the identification of the species of the tick. Additionally, the presence of DNA of Rickettsia amblyommatis was confirmed. This work is the first report of an exotic tick of the genus Amblyomma in a traveler from the US to Mexico and represents the second record of an imported tick attached to humans in Mexico.

Molecular detection of Coxiella-like endosymbionts and absence of Coxiella burnetii in Amblyomma mixtum from Veracruz, Mexico.

Ticks are obligate ectoparasites associated with a wide range of vertebrate hosts, including domestic animals. Moreover, ticks are capable of transmitting many pathogens such as Coxiella. To date, Coxiella burnetii, the etiological agent of coxiellosis or Q fever, is the only valid species of the genera. Nevertheless, a wide range of agents denominated Coxiella-like have been detected in recent studies, mainly associated with ticks. The pathogenicity of these Coxiella-like agents is controversial as some of them can infect both birds and humans. In Mexico, knowledge about Q fever is scarce and limited to historical serological records, and there is an overall lack of molecular proof of any agent of the genus Coxiella circulating in the country. Therefore, the aim of this study was to detect the presence of Coxiella in ticks associated with cattle in all 10 regions of Veracruz, Mexico. To accomplish this objective, first, we identified ticks collected from cattle and horses in Veracruz. Then, for Coxiella detection, DNA extraction from ticks and PCR amplification of the 16S-rDNA of Coxiella was performed. Finally, we performed a phylogenetic reconstruction to determine the Coxiella lineages detected. From the 10 regions sampled we collected 888 ticks grouped in 180 pools, and only five Amblyomma mixtum from the locality of Castán, and one from Los Angeles from Tuxpan were found positive, which represents a frequency of 20% for each locality. This study represents the first attempt at molecular detection of Coxiella in ticks associated with cattle in the state of Veracruz, the major livestock producer in the country. The findings of the present study are relevant as they establish a precedent regarding the circulation of Coxiella-like agents, as well as the absence in three municipalities of the state of Veracruz of C. burnetii, an abortive agent of livestock importance.

Rickettsial agents detected in the genus Psathyromyia (Diptera:Phlebotominae) from a Biosphere Reserve of Veracruz, Mexico.

Phlebotomine sand flies are considered the main vectors of Leishmania, the causal agents of leishmaniasis, which is a serious emerging public health problem worldwide. The use of biological control alternatives, like endosymbiotic bacteria (Wolbachia and Rickettsia), have been proposed to decrease sand fly populations and reduce Leishmania transmissions, yet only few records on the detection of Wolbachia or Rickettsia in sand flies are available worldwide. The aim of this study was to perform the molecular detection of Rickettsial agents associated with sand flies from the last patch of a rainforest in south-eastern Mexico, where a high prevalence of Leishmania infantum has been reported. Sampling effort of sand flies covered 300 trap-nights between 2011 and 2013, and a total of 925 specimens from twelve species were morphologically identified. Using PCR techniques, we identified a new lineage of the endosymbionts Rickettsia in Psathyromyia aclydifera (prevalence of 19.54%), and Wolbachia in Psathyromyia shannoni and Lutzomyia sp. (prevalence of 25%). The detected Wolbachia lineage was similar to the wWhi strain found in Pa. shannoni from Colombia and Nyssomyia whitmani from Brazil; whereas the identified Rickettsia represents a new lineage worldwide. This is the first record of Rickettsial agents associated to sand flies from this region, yet it remains for analysed if these bacteria possibly play a role as vector control agents, capable of reducing the sand fly populations in Mexico.

Influenza A H1N1 Virus 2009 Synthetic Hemagglutinin and Neuraminidase Peptides for Antibody Detection.

BACKGROUND: Influenza serologic diagnosis is mainly based on hemagglutination inhibition and microneutralization methods, both methods require handling living viruses under an enhanced biosafety level. AIM: The current study was performed for developing an ELISA using synthetic peptides to detect influenza A H1N1 virus 2009 specific antibodies in serum and saliva. METHODS: Alignments were made with H1N1 hemagglutinin and neuraminidase (HA and NA, respectively) sequences; only conserved sites were used for antigenicity prediction. Two synthetic peptides were assayed; one of neuraminidase (NA15) and one of hemagglutinin (HA-15) and used in ELISA for detecting IgG and IgA antibodies. A cross-sectional study was performed in three municipalities of Mexico City, using negative samples collected before the 2009 influenza outbreak, samples of people who became ill during the outbreak, and samples of the participants in the epidemiological study with or without symptoms. RESULTS: The determination of serum IgG antibodies with both peptides allowed differentiating between the post outbreak groups with respect to all others. No differences were found in IgA determination in saliva against both peptides. The frequency of positive participants for NA-15 was 9.5 and 8.8% for HA-15 in serum IgG; whereas the frequency of positive participants for NA-15 was 11%, and for HA-15 was 8.6% for saliva IgA. CONCLUSIONS: Synthetic peptides of the neuraminidase and hemagglutinin proteins can be used in ELISA for the determination of IgG and IgA antibodies against the influenza A H1N1 virus 2009.

What do studies in wild mammals tell us about human emerging viral diseases in Mexico?

Multiple species of viruses circulate in wild mammals, some of them potentially causing zoonosis. Most of the suspected viral zoonotic diseases affecting human patients remain unidentified with regard to their aetiological agent. The aim of this study is to summarize the state of knowledge of the viral richness associated with wild mammals in Mexico throughout 1900-2018 and their relationship with human cases. We compiled two databases, one of them containing all available published studies on potentially zoonotic viruses in wild mammals and another with human cases related to zoonotic viruses. The database on wild mammals covers the period of 1900-2018; the human case database spans 2000-2013. We calculated the richness of viral potential zoonotic agents and evaluated their geographical distribution. We found 262 records of 42 potential zoonotic viral species associated with 92 wild mammal species in 28 states across Mexico. Records of human viral cases were only found in 29 states, which did not overlap with the reports in wild mammals. We detected 25.6% (42/164) of viral zoonotic agents reported worldwide. This analysis opens a relevant topic of discussion for public health attention.