Genetic characterization of genes encoding the major surface proteins of Anaplasma marginale from cattle isolates in Thailand reveals multiple novel variants.

Bovine anaplasmosis is a serious tick-borne disease that is responsible for economic loss worldwide. The major surface proteins (MSPs), encoded by msp1 to msp5 genes of Anaplasma marginale, play an important role in host-pathogen and tick-pathogen interactions. These markers have been used for genetic characterization and phylogenetic studies. Despite domestic reports concerning suspected outbreaks of anaplasmosis in Thailand, genetic analysis of A. marginale in the country remains largely limited. Therefore, we aim to investigate the infection rate of the rickettsia organism in the Anaplasmataceae family throughout five regions of Thailand and to further characterize the key genetic markers: msp1a, msp2, and msp5 of A. marginale. From 2016 to 2021, we collected a total of 384 cattle blood samples across 18 provinces. Overall, the infection rate of the rickettsia organism in the Anaplasmataceae family was 46.1%. Over 65% of the positive samples were confirmed as A. marginale. We successfully obtained a total of 138 A. marginale msp1a (38), msp2 (79), and msp5 (21) sequences from all regions of the country. The msp1a and msp2 genes exhibit a high degree of genetic diversity, while the msp5 gene is highly conserved among the Thai isolates. Our findings regarding msp1a corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. Additionally, we found multiple novel variants for the first time in the current nationwide survey. We found 45 tandem repeat characters of the msp1a sequence. Among them, 24 characters were not shared with other countries. Collectively, we expanded the extent of genetic diversity in key markers; msp1a and msp2 genes, and further confirmed the previous finding that msp5 was highly conserved. The msp1a and msp2 genes could be useful for the surveillance of newly introduced strains. The current data may also be useful in designing a vaccine containing potential epitopes of different antigens in the future.

Liver- and Spleen-Specific Immune Responses in Experimental Leishmania martiniquensis Infection in BALB/c Mice.

Leishmania martiniquensis is a neglected cause of an emerging leishmaniasis in many countries, including France, Germany, Switzerland, the United States of America, Myanmar, and Thailand, with different clinical manifestations ranging from asymptomatic, cutaneous (CL), visceral (VL), and atypically disseminated CL and VL. The persistence of parasites and the recurrence of the disease after treatment are challenges in controlling the disease. To explore efficient prophylaxis and therapy, this study aimed to investigate infection outcome and organ-specific immune responses after inoculation with L. martiniquensis (MHOM/TH/2011/PG; 5 x 106 promastigotes) in BALB/c mice via intravenous and intraperitoneal routes. A quantitative PCR technique, targeting L. martiniquensis ITS1, was primarily established to estimate the parasite burden. We found that the infection in the liver resolved; however, persistent infection was observed in the spleen. Histopathology with Leishmania-specific immunostaining revealed efficient hepatic granuloma formation, while splenic disorganization with parasitized macrophages at different locations was demonstrated. The mRNA expression of Th1 cytokines (IFN-γ, TNF-α, IL-12p40) and iNOS in the liver and spleen was upregulated. In addition, high expression of IL-10 was observed in the spleen in the chronic phase, revealing a significant moderate correlation with the parasite persistence [r(12) = 0.72, P = 0.009]. Further clarification of the mechanisms of persistent infection and experimental infection in immunosuppressed murine models are warranted.

Natural infection of questing ixodid ticks with protozoa and bacteria in Chonburi Province, Thailand.

Ixodid ticks are important vectors of tick-borne disease agents affecting humans and animals, with wildlife often serving as important reservoirs. This study examined protozoal and bacterial infection in questing ticks in forest habitats in Chonburi Province, Thailand in 2015, using PCR and DNA sequencing techniques. A total of 12,184 ticks were morphologically identified to species and a subset of ticks were confirmed by PCR, targeting the tick mitochondrial 16S rRNA gene. Tick species collected included Haemaphysalis lagrangei (92.8%), H. wellingtoni (0.1%), and Rhipicephalus microplus (7.0%). In total, 419 tick pools [ELM(1] [ST2] were examined by PCR amplification of a fragment of the 18S rRNA gene of Babesia and Theileria species, and the 16S rRNA gene of bacteria in the family Anaplasmataceae. Results revealed a tick infection rate for the tick pools of 57.0% (239/419) including four pathogens and one bacterial symbiont. The highest infection rate in H. lagrangei, H. wellingtoni, and R. microplus pools was recorded for Anaplasma spp. at 55.6% (233/419) including three Anaplasma species genotype groups Anaplasma spp. closely related to A. bovis, A. platys, and unidentified Anaplasma spp. Theileria spp. showed a lower infection rate in H. lagrangei at 4.3% (18/419) with three Theileria species genotypes closely related to T. cervi, T. capreoli, and unidentified Theileria spp. Only 0.2% (1/419) of H. lagrangei pools contained Babesia spp., Ehrlichia spp., or Wolbachia spp. [ELM(3] [ST4] These findings provided information on tick species in wildlife habitats and detected protozoa and bacteria in the ticks. The results suggest that these tick species are possible vectors for transmitting tick-borne disease agents in Thailand wildlife habitats.