Ixodid ticks from wild and domestic animals in East and Central Asian flyways.

Tick-borne diseases have a significant impact on human and animal populations, posing an increasing threat to public health, particularly in the context of climate change. Along with the various natural hosts of ticks, birds play a notable role in transmitting ticks and tick-borne pathogens, indicating the importance of monitoring flyways and establishing a cooperative network for comprehensive surveillance and to collect diverse tick samples across various regions. This study aimed to develop an international network for surveillance of disease, collection of sufficient tick samples, and overall identification of the geographical distribution of host and ticks in Asian regions, especially in 11 countries on East Asian and Central Asian flyways. Ticks were collected from wild animals, domestic animals, and vegetation to identify the differences between Ixodid ticks and understand tick distribution. We collected a total 6,624 of ticks from 11 collaborating Asian countries, the Republic of Korea (ROK), Japan, Thailand, Philippines, Indonesia, Cambodia, Vietnam, Taiwan, Hong Kong, Mongolia and Pakistan. We identified 17 host animals and 47 species of both residential and migratory birds. Ticks from birds collected from four countries (ROK, Japan, Hong Kong and Mongolia) belonged to two genera, Haemaphysalis and Ixodes, including Haemaphysalis (H.) longicornis, H. flava, H. concinna, H. hystricis, H. formosensis, Ixodes (I.) nipponensis and I. persulcatus. The potential of migratory birds to cross ecological barriers with ticks and tick-borne diseases indicated the need for further investigations to understand the migration of birds as potential vectors and the new influx of zoonotic diseases along migratory bird flyways. This study suggests the potential risk of spreading tick-borne diseases through birds, thus highlighting the importance of international cooperative networking.

Analysis of the bacterial community in female Rhipicephalus microplus ticks from selected provinces in Luzon, Philippines, using next-generation sequencing.

Analysis of the tick microbiome can help understand tick-symbiont interactions and identify undiscovered pathogens, which may aid in implementing control of ticks and tick-borne diseases. The tropical cattle tick Rhipicephalus microplus is a widespread ectoparasite of cattle in the Philippines, negatively affecting animal productivity and health. This study characterized the bacterial community of R. microplus from Luzon, Philippines, through next-generation sequencing of 16s rRNA. DNA was extracted from 45 partially engorged female ticks from nine provinces. The DNA samples were pooled per province and then sequenced and analyzed using an open-source bioinformatics platform. In total, 667 operational taxonomic units (OTUs) were identified. The ticks in all nine provinces were found to have Coxiella, Corynebacterium, Staphylococcus, and Acinetobacter. Basic local alignment search tool (BLAST) analysis revealed the presence of known pathogens of cattle, such as Bartonella, Ehrlichia minasensis, and Dermatophilus congolensis. The tick samples from Laguna, Quezon, and Batangas had the most diverse bacterial species, whereas the tick samples from Ilocos Norte had the lowest diversity. Similarities in the composition of the bacterial community in ticks from provinces near each other were also observed. This is the first study on metagenomic analysis of cattle ticks in the Philippines, providing new insights that may be useful for controlling ticks and tick-borne diseases.

Molecular detection of Bartonella and Borrelia in pet dogs in Metro Manila and Laguna, Philippines.

Background and Aim: Bartonella and Borrelia are zoonotic vector-borne pathogens that can infect dogs and humans. Data on Bartonella and Borrelia in dogs in the Philippines are lacking. This study was conducted to validate previous reports and further investigate the occurrence of Bartonella and Borrelia spp. in cities of Metro Manila. Materials and Methods: A total of 182 canine blood samples were acquired with DNA using a commercial extraction kit from selected veterinary clinics in the cities of Metro Manila and Laguna, Philippines. The mammalian actin was amplified through polymerase chain reaction (PCR), followed by PCR assays targeting Bartonella gltA and Borrelia flaB. Further PCR assays targeting 16S of Borrelia and ospA and ospC of Borrelia burgdorferi were performed for those that showed flaB bands. Results: A positive band for gltA of Bartonella was observed in 9 (4.95%) samples, whereas a positive band for flaB of Borrelia was observed in 15 (8.24%) samples. Subsequent PCR assays for other genes of Borrelia were negative. Conclusion: These results confirmed the presence of Bartonella and warranted further investigation for the possible presence of other Borrelia species.

Molecular Detection and Characterization of Babesia and Theileria in Cattle and Water Buffaloes from Southern Luzon, Philippines.

Babesia and Theileria are tick-borne protozoan parasites that can cause significant economic losses in the cattle industry. This study aimed to contribute to the limited epidemiological data on Theileria orientalis as well as Babesia bigemina and B. bovis in large ruminants in the Philippines. Blood samples of 412 cattle and 108 water buffalo collected from four provinces in Southern Luzon, Philippines, were initially tested for the 18S rRNA gene of piroplasms through nested PCR. Positive samples were further subjected to species-specific PCR. The 18s rRNA of piroplasms was detected in 123 (29.9%) cattle and three (2.8%) water buffaloes. Theileria orientalis was found to be the most common piroplasm in cattle with a detection rate of 17.5%, followed by Babesia bovis and B. bigemina. Co-infections were also observed. Two water buffaloes were found infected with B. bovis, while one was positive for B. bigemina. The phylogenetic tree for B. bovis showed clustering of the isolates in two clades together with isolates from other countries, and a third separate clade. Meanwhile, the T. orientalis isolates in this study were distributed in three clades together with reported isolates from other countries. This study confirms the presence of T. orientalis in the Philippines and reports the genetic diversity of B. bovis and T. orientalis.

Molecular Prevalence of Anaplasma marginale and Ehrlichia in Domestic Large Ruminants and Rhipicephalus (Boophilus) microplus Ticks From Southern Luzon, Philippines.

Anaplasmosis and ehrlichiosis are tick-borne rickettsial diseases that cause significant economic losses in the livestock industry worldwide. Although bovine anaplasmosis is known to be endemic in the Philippines, epidemiological data is fragmented. Moreover, little is known about bovine ehrlichiosis in the country. In this study, the prevalence of Anaplasma marginale and Ehrlichia in cattle and water buffalo from provinces in the southern part of Luzon, Philippines, was investigated through PCR. Blood samples from 620 animals comprised of 512 cattle and 108 water buffalo and 195 tick samples were subjected to nested PCR targeting the groESL gene of Anaplasmataceae. Positive samples were further subjected to another nested PCR and conventional PCR to amplify the A. marginale groEL gene and the Ehrlichia dsbA gene, respectively. Selected A. marginale-positive samples were also subjected to nested PCR targeting the msp5 gene. Regardless of the animal host, the overall prevalence in blood samples obtained was 51.9% for Anaplasmataceae, 43% for A. marginale, and 1.1% for Ehrlichia. No water buffalo were positive for Ehrlichia. Meanwhile, 15.9, 6.7, and 2% of the tick samples, all morphologically identified as Rhipicephalus (Boophilus) microplus, were positive for Anaplasmataceae, A. marginale, and Ehrlichia, respectively. Sequence analysis of selected A. marginale msp5 amplicons showed that the isolates from the region share 94-98% identity to reported A. marginale from other countries. The phylogenetic tree showed clustering of isolates in the region and a close relationship with A. marginale isolates from other countries. Sequences of Ehrlichia amplicons from cattle and ticks were 97-100% similar to reported Ehrlichia minasensis isolates. This study showed the high prevalence of A. marginale in Luzon, Philippines, and provided the first molecular evidence of E. minasensis in the country.

Determination of resistance status to amitraz in the cattle tick Rhipicephalus (Boophilus) microplus from Luzon, Philippines, through bioassay and molecular analysis.

Tick infestations and tick-borne diseases are among the leading causes of economic losses in the cattle industry worldwide. Amitraz is one of the most commonly used chemical acaricides against the cattle tick Rhipicephalus (Boophilus) microplus. Resistance to amitraz has been reported in many countries but not in the Philippines. This study aimed to determine whether cattle ticks from provinces in northern and southern Luzon, Philippines, are resistant to amitraz. Engorged or nearly engorged female ticks were collected from 21 farms and allowed to lay eggs to produce larvae. Larval packet test (LPT) was performed using three concentrations of amitraz, as well as a negative control (diluent). Reverse-transcription polymerase chain reaction (RT-PCR) was also performed to amplify a fragment of the octopamine/tyramine receptor gene and was subjected to sequence analysis by multiple nucleotide and amino acid sequence alignments together with reference strain, amitraz-susceptible strain, and amitraz-resistant strain reported in other countries. LPT results suggest the absence of resistance in all of the tested populations. Interestingly, analysis of the octopamine/tyramine receptor amino acid sequence revealed four out of 14 larval pools having substitutions similar to that of the reported amitraz-resistant strains. Although no apparent resistance was observed in this study, prudent use of amitraz should be practiced as the development of resistance is still likely to occur in the future.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting the Citrate Synthase Gene for Detection of Ehrlichia canis in Dogs.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples-after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar's test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.

Molecular Detection of Rickettsia Spp. and Coxiella Burnetii in Cattle, Water Buffalo, and Rhipicephalus (Boophilus) Microplus Ticks in Luzon Island of the Philippines.

Rickettsia and Coxiella burnetii are zoonotic, tick-borne pathogens that can cause febrile illnesses with or without other symptoms in humans, but may cause subclinical infections in animals. There are only a few reports on the occurrence of these pathogens in cattle and water buffalo in Southeast Asia, including the Philippines. In this study, molecular detection of Rickettsia and C. burnetii in the blood and in the Rhipicephalus (Boophilus) microplus ticks of cattle and water buffalo from five provinces in Luzon Island of the Philippines was done. A total of 620 blood samples of cattle and water buffalo and 206 tick samples were collected and subjected to DNA extraction. After successful amplification of control genes, nested PCR was performed to detect gltA of Rickettsia and com1 of C. burnetii. No samples were positive for Rickettsia, while 10 (cattle = 7, water buffaloes = 3), or 1.6% of blood, and five, or 1.8% of tick samples, were C. burnetii-positive. Sequence analysis of the positive amplicons showed 99-100% similarity to reported C. burnetii isolates. This molecular evidence on the occurrence of C. burnetii in Philippine ruminants and cattle ticks and its zoonotic nature should prompt further investigation and surveillance to facilitate its effective control.

Molecular detection of tick-borne pathogens in canine population and Rhipicephalus sanguineus (sensu lato) ticks from southern Metro Manila and Laguna, Philippines.

BACKGROUND: The tropical climate of the Philippines and the high population of dogs, particularly in cities, favors the life-cycle of the brown dog tick, Rhipicephalus sanguineus (sensu lato), a vector of several canine tick-borne pathogens (TBPs) including zoonotic Rickettsia spp. Suspected cases of infections are commonly encountered in veterinary clinics, but the specific TBPs are rarely identified. Furthermore, infection with Rickettsia is not being clinically examined in dogs. In this study, the occurrence of TBPs in blood and ticks collected from household and impounded dogs in highly populated areas of the Philippines, Metro Manila, and the nearby province of Laguna, was examined. RESULTS: A total of 248 blood samples and 157 tick samples were subjected to PCR. First, samples were screened using primers for Anaplasma/Ehrlichia spp. and Babesia/Hepatozoon spp. Those that turned positive were further subjected to species-specific PCR. Rickettsia spp. were also detected through a nested PCR. Of the 248 blood samples, 56 (22.6%) were positive for Anaplasma/Ehrlichia spp., while 19 (7.6%) were positive for Babesia/Hepatozoon spp. Species-specific PCR revealed that 61 (23.4%) had a single TBP, with Ehrlichia canis being detected in 39 (15.7%) dogs, while 14 (5.6%) dogs were positive for different combinations of two to four TBPs. Rickettsia infection was detected in 6 (2.4%) dogs. In tick samples, 8 (3.2%) were positive for Ehrlichia/Anaplasma spp., while only 1 (0.63%) was positive for Babesia/Hepatozoon spp. As in the blood samples, E. canis was the most detected, being found in 5 (2%) samples. No tick samples tested positive for Rickettsia spp. CONCLUSION: Ehrlichia canis is the most common TBP affecting dogs in the Philippines. Co-infection with TBPs is quite common, hence testing for multiple TBPs is necessary. Through nested PCR, Rickettsia infection was detected in dogs, and to the authors' knowledge, this study provides the first molecular evidence of Rickettsia infection in dogs in the Philippines.