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.

Zoonotic transmission of intestinal helminths in southeast Asia: Implications for control and elimination.

Intestinal helminths are extremely widespread and highly prevalent infections of humans, particularly in rural and poor urban areas of low and middle-income countries. These parasites have chronic and often insidious effects on human health and child development including abdominal problems, anaemia, stunting and wasting. Certain animals play a fundamental role in the transmission of many intestinal helminths to humans. However, the contribution of zoonotic transmission to the overall burden of human intestinal helminth infection and the relative importance of different animal reservoirs remains incomplete. Moreover, control programmes and transmission models for intestinal helminths often do not consider the role of zoonotic reservoirs of infection. Such reservoirs will become increasingly important as control is scaled up and there is a move towards interruption and even elimination of parasite transmission. With a focus on southeast Asia, and the Philippines in particular, this review summarises the major zoonotic intestinal helminths, risk factors for infection and highlights knowledge gaps related to their epidemiology and transmission. Various methodologies are discussed, including parasite genomics, mathematical modelling and socio-economic analysis, that could be employed to improve understanding of intestinal helminth spread, reservoir attribution and the burden associated with infection, as well as assess effectiveness of interventions. For sustainable control and ultimately elimination of intestinal helminths, there is a need to move beyond scheduled mass deworming and to consider animal and environmental reservoirs. A One Health approach to control of intestinal helminths is proposed, integrating interventions targeting humans, animals and the environment, including improved access to water, hygiene and sanitation. This will require coordination and collaboration across different sectors to achieve best health outcomes for all.

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.

Giardia duodenalis in Captive Tigers (Panthera tigris), Palawan Bearcats (Arctictis binturong whitei) and Asian Palm Civet (Paradoxurus hermaphroditus) at a Wildlife Facility in Manila, Philippines.

BACKGROUND: This study was conducted to determine the prevalence of Giardia duodenalis in captive animals in a wildlife facility. This is the first study conducted in these animals from the facility. METHODS: Eight captive tigers (Panthera tigris), two Palawan bearcats (Arctictis binturong whitei) and one Asian Palm Civet (Paradoxurus hermaphroditus) currently housed at a wildlife facility in Manila, Philippines were considered in 2012. These animals were apparently healthy with no signs of disease during the study. Sample collection was done twice at two months interval where freshly voided fecal samples were grossly examined, characterized and preserved in Sodium Acetate Formalin (SAF). The samples were used to determine the presence of G. duodenalis using modified flotation-sedimentation and commercially available immuno-chromatographic assay test kit. RESULTS: All fecal samples tested were negative for the presence of G. duodenalis trophozoites, and cysts using the former. Furthermore, none of the samples tested positive for and G. duodenalis antigen using immune-chromatographic assay. CONCLUSION: There is no existing infection of G. duodenalis among captive tigers, Palawan Bearcats and Asian palm civet housed at the wildlife facility.

Molecular identification and prevalence of Dictyocaulus spp. (Trichostrongyloidea: Dictyocaulidae) in Swedish semi-domestic and free-living cervids.

Lungs of 102 roe deer (Capreolus capreolus), 136 moose (Alces alces), 68 fallow deer (Dama dama), and six red deer (Cervus elaphus) were examined during hunting seasons from 16 September 1997 to 1 March 2000. The aim was to determine the species composition and prevalence of Dictyocaulus lungworms in these hosts in Sweden. Worms were identified following polymerase chain reaction (PCR) amplification of the internal transcribed spacer of ribosomal DNA (ITS2), followed by hybridization with four species-specific oligonucleotides. In addition, 50 lungworms from five reindeer (Rangifer tarandus) from Norway were similarly analyzed. A total of 399 worms were recovered and analyzed representing a range of 29-128 worms per host species. All specimens from roe deer were identified as Dictyocaulus capreolus, whereas those from red deer and reindeer were identical with D. eckerti. From moose, 73 (81.1%) of the worms were identified as D. capreolus whereas 17 (18.9%) were D. eckerti. The ITS2 sequence of fallow deer lungworms differed significantly when compared with the ITS2 of D. viviparus, D. capreolus, and D. eckerti. This indicated that fallow deer in Sweden may be infected with a new genotype of Dictyocaulus spp. Consequently, a specific probe designed for the ITS2 from this Dictyocaulus sp. hybridized exclusively with samples from lungworms of fallow deer. Interestingly, no D. viviparus were found in any of these hosts. The prevalence of infection in each host was as follows: D. capreolus in roe deer (14.7%) and moose (10.6%); D. eckerti in moose (0.7%) and red deer (33.3%); and Dictyocaulus sp. in fallow deer (10.3%). Regardless of lungworm species, the overall prevalence of Dictyocaulus spp. in these hosts was 12.2%. Prevalence between male and female animals and among the different age groups did not differ significantly. Finally an enzyme linked immunosorbent assay (ELISA) specific for patent D. viviparus infection in cattle was utilized to analyze lung tissue fluids from infected animals. All samples from roe deer, red deer, and fallow deer were negative in the ELISA. However, three out of twelve (25%) samples from moose and 17 of 40 (43%) samples from cattle were positive. This indicated that moose anti-D. capreolus antibodies recognized the D. viviparus antigen and that anti-cattle immunoglobulin cross-reacted with moose antibodies.