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Tick-borne diseases (TBD) cause enormous losses for farmers. Backyard raising comprises majority of the livestock population in the Philippines, but TBD information in backyard livestock is scarce. In this study, 48 cattle and 114 water buffalo samples from Quezon province, Philippines were molecularly screened for tick-borne pathogens. Anaplasma marginale (16.67%) and hemoplasma (20.99%) were detected in the samples. A. marginale infection (P=0.0001) was significantly higher in cattle, while hemoplasma infection (P=0.011) was significantly higher in water buffaloes. A. marginale isolates from this study were highly similar to previous isolates from the Philippines while Mycoplasma wenyonii and Candidatus Mycoplasma haemobos were the identified hemoplasma species. Our findings reveal additional information on the TBD situation of Philippine backyard livestock.

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4–99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.

Toxoplasmosis is a widely distributed zoonotic infection caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii. It is mainly transmitted through the ingestion of oocysts shed by an infected cat acting as its definitive host. The key to effective control and treatment of toxoplasmosis is prompt and accurate detection of T. gondii infection. Several laboratory diagnostic methods have been established, including the most commonly used serological assays such as the dye test (DT), direct or modified agglutination test (DAT/MAT), indirect hemagglutination test (IHA), latex agglutination test (LAT), indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assays (ELISA), immunochromatographic tests (ICT), and the western blot. Nonetheless, creating specific and reliable approaches for serodiagnosis of T. gondii infection, and differentiating between acute and chronic phases of infection remains a challenge. This review provides information on the current trends in the serodiagnosis of human toxoplasmosis. It highlights the advantages of the use of recombinant proteins for serological testing and provides insight into the possible future direction of these methods.

Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.

Bovine babesiosis represents a serious threat to the cattle industry in the tropics and subtropics. Although several Babesia species infect cattle, only B. bovis, B. bigemina and B. divergens are known to cause clinical babesiosis. However, our recent study demonstrated that the newly discovered Babesia sp. Mymensingh might be a virulent species capable of causing clinical babesiosis in cattle. The objective of this study was to determine the host range and geographical distribution of Babesia sp. Mymensingh on a global scale. A total of 2,860 archived DNA samples from 2,263 cattle in Sri Lanka (n = 672), the Philippines (n = 408), Vietnam (n = 460), Uganda (n = 409), Brazil (n = 164) and Argentina (n = 150); 419 buffalo in Sri Lanka (n = 327) and Vietnam (n = 92); and 127 goats and 51 sheep in Vietnam were screened using a Babesia sp. Mymensingh-specific PCR assay. Babesia sp. Mymensingh infection was detected in cattle, buffalo, sheep and goats. Cattle of all countries surveyed in this study except Brazil were found to be infected with Babesia sp. Mymensingh. The highest positive rates were recorded in cattle from the Philippines (11.3%) and Vietnam (9.6%), followed by Argentina (4.7%), Sri Lanka (1.5%) and Uganda (1.0%). Buffalo were found to be infected with this parasite in Sri Lanka (1.2%) and Vietnam (10.9%). Unexpectedly, Babesia sp. Mymensingh was also detected in sheep (2.0%) and goats (1.3%) from Vietnam. These findings were confirmed by PCR amplicon sequencing. In conclusion, our present findings indicate that Babesia sp. Mymensingh, which infects cattle, buffalo, sheep and goats, is endemic in Asia, Africa and South America.

Background Gastrointestinal (GIT) parasites can affect poultry productivity by compromising its health. It is well studied in other countries, but the documented reports in the Philippines have been limited. Aim The aim of the present study was to evaluate the presence of GIT parasites in selected small-scale poultry layer farms in Leyte, Philippines. Materials and methods A total of 243 stool samples from eight small-scale poultry layer farms in Leyte, Philippines, were examined for GIT parasites using floatation and sedimentation technique. Profile parameters were also obtained. Fecal samples were collected and analyzed using floatation and sedimentation techniques. Statistical significance between GIT parasite positivity and profile parameters was determined using Chi-square test. Results GIT parasites were detected in 92.2% of the samples (24.7% with single infection, 42.0% with 2-3 parasites, and 25.5% with three or more parasites). The common parasites detected were Ascaridia spp. (41.2%), Heterakis spp. (59.3%), Capillaria spp. (10.7%), Eimeria spp. (43.2%), and Strongyloides spp. (74.1%). Some profile parameters, including farm location, years in business, number of workers, nearby water system, the practice of fecal cleaning, and presence of other animals, were found to be significantly associated with GIT positivity. Conclusion GIT parasites were detected in the poultry of small-scale layer farms in selected areas in Leyte, Philippines. This finding calls for the importance of routine GIT parasite monitoring and the implied need for regular deworming or dewormer rotation in the area.

Theileria equi and Babesia caballi are tick-borne protozoan parasites that can cause anemia in horses. In the Philippines, serological detection of these parasites has only been reported in the Northern area (Luzon). In this study, 105 horses from Cebu and Bohol, Philippines were tested using peripheral blood smear examination (PBSE), immunochromatographic test (ICT) strips, and PCR. Clinical history, presenting clinical signs and complete blood count were obtained. Results revealed that although all horses were negative using PBSE, 23 (21.9%) were positive (12 for T. equi, and 11 for B. caballi) using ICT. PCR revealed 26 and 2 horses positive for T. equi and B. caballi, respectively. All positive horses showed no clinical signs. Partial DNA sequences of representative amplicons were found 100% identical to GenBank registered T. equi and B. caballi sequences. Statistical analyses revealed that location was found associated with T. equi PCR positivity and B. caballi seropositivity. This study documents the first serological detection of T. equi and B. caballi in horses in the southern area of the Philippines, and their first molecular detection and characterization in the country.

Tick-borne diseases (TBDs) remain to be a global animal health threat. Developing countries like the Philippines is not exempt to this. Despite the potential impact TBDs can give to these countries, local government initiatives and researches remain to be limited. In the Philippines, most epidemiological studies were confined only to specific areas, and predominantly in the Northern Area. Due to its unique geography and limited studies, the current nationwide status of most TBDs could not be clearly established. This review mainly covered published studies and presented challenges in the conduct of TBD research in the Philippines, which may be similar to other Southeast Asian or developing countries. To date, reported livestock TBD pathogens in the Philippines include Anaplasma, Babesia, Theileria, and Mycoplasma spp. With the ubiquitous presence of the Rhipicephalus microplus ticks in the country, it is highly probable that other pathogens transmitted by these vectors could be present. Despite studies on different TBDs in the livestock sector, the Philippine government has not yet heightened its efforts to implement tick control measures as part of the routine animal health program for local farmers. Further studies might be needed to determine the nationwide prevalence of TBDs and the presence of other possible tick species and TBD pathogens. The Philippine scenario may present situations that are similar to other developing countries.

Background: Ehrlichia, Anaplasma, and Babesia spp. are canine pathogens transmitted by the Rhipicephalus sanguineus tick which can cause varied clinical signs. These pathogens have been investigated in the Philippines, but coinfection has not been reported yet. Aim: The aim of this study was to evaluate the presence of Ehrlichia/Anaplasma and Babesia spp. in Philippine dogs. Materials and Methods: A total of 100 dogs from seven different veterinary establishments in Cebu, Philippines, were examined for Ehrlichia/Anaplasma and Babesia spp. infection using peripheral blood smear examination and polymerase chain reaction (PCR). Inclusion criteria included a history or presence of tick infestation, anemia, and/or thrombocytopenia. Clinical signs were recorded. Statistical analyses were performed between PCR positivity and clinical signs and hematological results. Results: A total of 10 and 18 dogs were found to be positive for Ehrlichia/Anaplasma and Babesia spp., respectively. One animal was PCR positive for both pathogens, which is the first report of coinfection in the country. The most common clinical signs observed include inappetence (89%), lethargy (80%), thrombocytopenia (85%), and anemia (74%). Analyses revealed that inappetence (p=0.044) and weight loss (p=0.028) were found statistically significant with Ehrlichia/Anaplasma infection. Basophil (p=0.001) and eosinophil counts (p=0.000) were also found significantly different between Ehrlichia/Anaplasma spp.-positive and -negative dogs. On the other hand, differential monocyte count (p=0.009) was found significantly different between Babesia spp.-positive and -negative dogs. Conclusion: The present study showed low infection rates of canine ehrlichiosis/anaplasmosis and babesiosis and provided additional evidence for the presence of the pathogens in the area.

Tick-borne diseases (TBDs) considerably impair equine health and productivity. Moreover, TBDs, particularly equine piroplasmosis, impede international movement and trade of equids, which is a vital component of the global horse racing industry. In the Philippines, horse racing is a lucrative industry generating millions of USD annually. However, information on equine TBDs is scarce. This study intended to describe molecularly the equine tick-borne infections in a racehorse park in Cavite, Philippines and identify the risk factors associated with the infections. One hundred twenty-four (n = 124) thoroughbred racehorses were sampled and screened for selected tick-borne protozoan and bacterial pathogens using polymerase chain reaction (PCR) assays. Racehorses were positive for Babesia caballi (12.10%, 15/124), Theileria equi (0.81%, 1/124), Anaplasma phagocytophilum (10.48%, 13/124), Borrelia burgdorferi sensu lato (38.71%, 48/124), A. marginale (0.81%, 1/124), and Coxiella burnetii (0.81%, 1/124). Rickettsia was not detected in the samples. Gender was determined as a significant risk factor for B. caballi infection. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63–100% identity with each other and were classified as genotype A. Meanwhile, the sequence obtained from the lone T. equi-positive sample was 99.77% identical to isolates from Spain, Switzerland, China, Saudi Arabia, and South Korea, and was confirmed as genotype E based on the 18S rRNA gene. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis. Partial sequences of Borrelia 5–23S rRNA were most closely related to Bo. japonica and other Borrelia sp. isolates from various countries. This study reports the first molecular detection of Borrelia and Anaplasma and the identification of B. caballi and T. equi genotypes in racehorses in the Philippines. Findings from this study shall be useful in crafting equine tick and TBD control and prevention programs in the country.

Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.

application/pdf, Anaplasma species of the family Anaplasmataceae, order Rickettsiales are tick-borne organisms that can cause disease in animals and humans. In Japan, all recognized species of Anaplasma (except for Anaplasma ovis) and a potentially novel Anaplasma sp. closely related to Anaplasma phagocytophilum have been reported. Most of these detected tick-borne pathogens are believed to be lowly pathogenic in animals in Japan although the zoonotic A. phagocytophilum has recently been reported to cause clinical signs in a dog and in humans. This review documents the studies and reports about Anaplasma spp. in Japan. c Ybanez and Inokuma.

Despite the large population of stray dogs in Thailand, there is limited information on the prevalence of canine vector-borne diseases (CVBDs). In this study, a molecular survey was conducted to determine the prevalence of Babesia spp., Ehrlichia canis, Hepatozoon spp., Anaplasma platys and Mycoplasma spp. in dogs in Thailand. Of the 181 dog blood samples tested by PCR, 78/181 (43.1%) were found to be infected with one or more pathogens. The overall prevalence rates of Mycoplasma spp., Hepatozoon spp., Babesia spp., A. platys and E. canis infections were 19.9%, 18.8%, 9.4%, 4.4% and 3.9%, respectively. To the authors' knowledge, this is the first report of Mycoplasma infection in Thailand in dogs. The current findings are important for future surveillance of CVBDs and designing appropriate approaches for diagnosis and control for the diseases in Thailand.

The increasing number and severity of surra outbreaks in the Philippines led the government to consider it as the second most important disease of livestock in the country. It is one of the most economically important animal parasitic diseases and has been reported in several animal species, including water buffaloes, cattle, and horses in different regions of the Philippines. However, it has not yet been reported in Cebu, the usual gateway of livestock trade in the area that raises 6% of the 3.75 million goats in the country. In the current study, a PCR-based assay was conducted for the molecular detection and characterization of Trypanosoma evansi in goats in Cebu. A total of 251 goats were randomly sampled from four farms. DNA was extracted and ITS1-PCR was applied to detect different trypanosomes in goats. Eighty-five out of the 251 (33.9%) samples tested positive for T. evansi, two of which were also positive for T. theileri-like trypanosome. The detection rate of T. evansi was slightly higher in male goats (38.3%) than in females (32.5%), and in younger goats (34.5%) than in adults (33.5%). The findings, however, did not differ significantly to suggest any association between sex and age with T. evansi infection in goats. The detection of T. evansi and T. theileri-like trypanosome in goats was confirmed by sequence analysis of ITS1 region. To our knowledge, this is the first report on the molecular detection and identification of caprine T. evansi infection in Cebu, Philippines.

The Tropical Council for Companion Animal Parasites Ltd. (TroCCAP) is a not-for-profit organisation whose mission is to independently inform, guide and make best-practice recommendations for the diagnosis, treatment and control of companion animal parasites in the tropics and sub-tropics, with the aim of protecting animal and human health. In line with this primary mission, TroCCAP recently developed guidelines for the diagnosis, treatment and control of feline and canine parasites in the tropics. The development of these guidelines required unique and complex considerations to be addressed, often inapplicable to developed nations. Much of the tropics encompass middle-to-low income countries in which poor standards of environmental hygiene and large populations of stray dogs and cats coexist. In these regions, a range of parasites pose a high risk to companion animals, which ultimately may place their owners at risk of acquiring parasitic zoonoses. These considerations led to the development of unique recommendations with regard, for example, to deworming and endoparasite testing intervals for the control of both global and 'region-specific' parasites in the tropics. Moreover, the 'off-' or 'extra'-label use of drugs for the treatment and control of parasitic infections is common practice in many tropical countries and many generic products lack manufacturers' information on efficacy, safety, and quality control. Recommendations and advice concerning the use of such drugs and protocols are also addressed in these guidelines. The formation of these guidelines is an important first step towards improving the education of veterinarians specifically regarding best-practice for the diagnosis, treatment and control of canine and feline parasites in the tropics.

Background: Tick-borne diseases are caused by a wide variety of viruses, pathogens, and diseases. Anaplasma, Ehrlichia, and Babesia spp. are among the most known tick-borne pathogens in Asia. In the Philippines, these pathogens were already reportedly present in dogs and large ruminants, but no study has been reported yet evaluating their presence in goats. Aim: The present study aimed to evaluate the presence of Anaplasma, Ehrlichia, and Babesia spp. in goats in Cebu, the Philippines. Materials and Methods: A total of 100 blood samples from goats were collected in Cebu, the Philippines. Profile of sampled goats including age, body score, and sex was obtained. Peripheral blood smear examination and DNA extraction were performed. Nested polymerase chain reaction assay was used to evaluate the presence of Anaplasma, Ehrlichia, and Babesia spp. Results: None of the samples were found positive with Anaplasma, Ehrlichia, and Babesia spp. infection. Conclusion: Tested goats were negative with Anaplasma, Ehrlichia, and Babesia spp. and calls for continuous surveillance of these pathogens due to the reported detection of these pathogens in other livestock animals in the area.

application/pdf, Toxoplasma gondii is a single-celled intracellular apicomplexan parasite that causes toxoplasmosis. It is capable of infecting humans and nearly all warm-blooded animals including pigs, but cats are the only known definitive host. This ubiquitous zoonotic pathogen can cause abortion, stillbirth and fetal abnormalities, and has been associated with mental and behavioral changes in humans. Acute infection is potentially fatal in immunocompromised individuals. The present study aimed to assess the Toxoplasma seroprevalence in pigs, humans and cats after its initial reported detection in pigs about three decades ago in Cebu, Philippines. A total of 924 humans, 104 cats and 514 slaughter pigs were tested for antibodies against T. gondii using a commercial latex agglutination test. The results revealed positive detection rates of 26.3% (244/924) for humans, 42.3% (44/104) for cats and 13.4% (69/ 514) for slaughter pigs. Statistical analyses revealed that the area (P = 0.004), cat ownership (P = 0.020), the frequency of contact with cats (P < 0.0001) and consumption of street foods (P = 0.043) were significantly associated with seropositivity for T. gondii in humans. Meanwhile, the use of litter trays (P = 0.001) and contact with other animals (P = 0.007) were significantly associated with seropositivity in cats. The odds ratio for selected significant factors revealed that living in suburban areas (OR 1.66, 95% CI: 1.20–2.31), owning a cat (OR 1.482, 95% CI: 1.07–2.07) and eating street foods (OR 1.585, 95% CI: 1.01–2.48) were associated with an increased risk of T. gondii exposure in humans. In cats, the use of a litter tray (OR 4.5, 95% CI: 1.73–11.71) was associated with an increased risk of exposure. None of the profile parameters were found to be significantly associated with seropositivity in slaughter pigs (P > 0.05). This study is the first report of the serological detection of T. gondii in humans and cats in Cebu, Philippines, and the first assessment of the prevalence of the parasite in pigs in the area since its initial detection in 1982. This is also the first report documenting the seropositivity of T. gondii in pregnant women in the country. The confirmed seropositivity of T. gondii in Cebu, Philippines, in the present study implies the endemicity of toxoplasmosis in this area and highlights the need for routine testing and increased public awareness. © 2019 Ybañez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background: Gastrointestinal tract (GIT) parasites affect the health of dogs and may also be zoonotic. The prevalence of these parasites has been well studied in several countries, but reports in the Philippines have been limited. Aim: This study generally aimed to detect the presence of common GIT parasites in owned and shelter dogs in Cebu, Philippines. Materials and Methods: A total of 200 fecal samples (130 from owned dogs and 70 from shelter dogs) were collected. Profiles of owned dogs and their owners were obtained. Fecalysis was performed using three methods: Direct smear, sedimentation, and flotation techniques. Results: Majority of the sampled dogs were 5 years old and below that (79.2%), male (64.6%) and of pure breed (53.1%). Among the most common parasites detected were Ancylostoma, Trichuris and Toxocara spp. Statistical analyses revealed a significant association between the presence of parasites and the body score of the dogs (p=0.000), the deworming status (p=0.000), and the rearing practice (contact with other dogs, p=0.000, where it spends its time (p=0.000), plays in the grass (p=0.050), where it defecates (p=0.014), contact with other animals (p=0.000). Conclusion: GIT parasites were detected in owned and shelter dogs in Cebu, Philippines. The results of this study can serve as baseline information about the canine parasitic fauna in the Philippines.

Hemoplasmosis caused by Mycoplasma spp. have been associated with major economic losses in the global dairy production. Hemoplasma studies in the Philippines are limited despite its potential impact. This study mainly aimed to detect the presence of hemoplasma species in dairy water buffaloes and cattle and know their ectoparasite biodiversity in Bohol, Philippines. Detection of Mycoplasma spp. was performed using peripheral blood smear examination (PBSE) and standard PCR using whole blood samples collected from 100 dairy water buffaloes and 40 dairy cattle. Available records on the average annual, monthly and daily milk production were compared between PCR-positive and PCR-negative animals. Ectoparasites were manually collected and identified. While PBSE results were all negative, PCR testing showed that 80% (80 water buffaloes and 32 cattle) were positive for Mycoplasma spp. On the other hand, a total of 1436 ectoparasites were collected (609 Haematopinus and 827 Rhipicephalus spp.). DNA sequencing revealed that obtained sequences (193 bp) from 7 animals were 99.5 to 100% similar to registered Mycoplasma wenyonii sequences. The study reports the first molecular characterization of M. wenyonii in the Philippines and probably the first detection in dairy water buffaloes in Southeast Asia.

Aim: The study aimed to document the clinical and hematological observations of dogs found serologically positive for Ehrlichia canis and to identify parameters or factors that are associated with the disease with focus on the anemic and thrombocytopenic state of the infected dogs. Materials and Methods: From 7 participating veterinary establishments, a total of 913 cases from 2003 to 2014 were initially assessed using inclusion criteria, including E. canis diagnosis by the attending veterinarian and the presence of ticks or history of infestation, thrombocytopenia, and/or anemia. From these, 438 cases that were found serologically positive for E. canis using commercial test kits were selected. Profile, clinical observations and hematological test results were obtained from the selected cases. Computations for statistical associations between the anemic and thrombocytopenic state of the infected dogs and their profile, observed clinical signs and other hematological values were performed. Results: Most of the dogs were purebred (60.0%) and female (51.1%) and were within the age range of 1-5 years (38.4%). The mean packed cell volume (PCV), red blood cell (RBC) count, and platelet count were lower than the normal values while the absolute count of basophils were higher than normal values. Creatinine and blood urea nitrogen (BUN) appear to be elevated. The most common clinical signs observed were inappetence (41.3%), lethargy/depression (35.6%), vomiting (32.4%), fever (18.5%), paleness (8.2%), and epistaxis (6.6%). Analyses showed that there were no significant differences on the hematological values and clinical signs between thrombocytopenic and non-thrombocytopenic seropositive dogs. Moreover, very weak correlations between platelet count and RBC count, absolute lymphocyte count, and neutrophil count were found. On the other hand, only paleness (p=0.008) and epistaxis (p=0.004) were found to be significantly different between anemic and non-anemic patients. This coincided with the linear regression results where PCV (p=0.000, R=0.787, R2=0.619) was moderately correlated with the RBC count. In addition, eosinophil count was found weakly correlated. Conclusion: E. canis infection in dogs may produce varied clinical signs that may be influenced by the thrombocytopenic and anemic states of affected animals. Complete blood counts remain important in the diagnosis of the disease, especially the platelet and RBC counts. Creatinine, BUN and alanine aminotransferase can be of value in the diagnosis of the infection. Several cases were lost to follow-up and appeared to be a challenge for handling veterinarians to monitor compliance of owners and progress of infected patients.

application/pdf, Aim: Anaplasma platys, the causative agent of infectious canine cyclic thrombocytopenia, is a tick-borne pathogen that also has been implicated as potentially zoonotic. To provide molecular evidence on the multiple infections of A. platys variants in Philippine dogs. Materials and Methods: DNA fragments of A. platys from infected dogs in the Philippines were molecularly characterized. For screening, 25 dogs suspected to have canine anaplasmosis were tested using a 16S rRNA-based nested polymerase chain reaction (PCR). Infection was confirmed by sequencing of positive amplicons. Second round PCR targeting a longer 16S rRNA fragment was subsequently performed on the first round PCR amplicons of the positive samples. Further characterization using the heat-shock operon (groEL) gene was also performed on the A. platys-positive samples. Results: 10 16S rRNA sequences were obtained and found 99.6-100% identical to each other and 99.6-99.7% identical to the closest registered A. platys sequences. On the other hand, 36 groEL clone sequences were obtained and found to be 85.1-99.8% identical with each other and 85.0-88.9% identical to the closest previously registered A. platys sequences. Four dogs were found coinfected with 2-3 groEL variant sequences. Phylogenetic analyses suggest that the detected A. platys in the Philippines may represent unique variants. Conclusion: A. platys variants were detected in Philippine dogs. Coinfection of different A. platys variants in dogs was also demonstrated. The present study may indicate the potential genetic diversity of A. platys in the country.

A total of 658 cattle in 6 provinces in the Philippines were screened for Anaplasma marginale infection by using a diagnostic heat-shock operon (groEL) gene-PCR assay. The screening-positive samples were further tested using the major surface antigen protein 1a (Msp1a) gene-PCR assay. Screening PCR results showed 130 cattle (19.8%) were positive for the A. marginale infection. Subsequent amplification using the Msp1a gene only showed 93 samples (14.1%) to be positive. In addition, 37 tandem-repeat structures, including 20 novel structures, and 41 distinct genotypes were identified. Interestingly, multiple infections of 4 different genotypes were also observed in A. marginale-infected cattle. The present study demonstrated the prevalence and characterization of diverse genotypes of A. marginale in the Philippine cattle.

The present study aimed to determine the complete citrate synthase (gltA) and heat-shock protein (groEL) gene sequences of Anaplasma bovis and to infer phylogenetic relationships within the genus Anaplasma. Multiple alignments from single and concatenated sequences of the 16S rRNA, gltA and groEL genes of the genus Anaplasma were subjected to phylogenetic analyses. Percent identities of A. bovis nucleotide sequences were found highest with A. phagocytophilum in gltA (65.4%) and groEL (79.8%). Single gene phylogenetic tree results assumed similar phylogenetic positions within the genus Anaplasma, except for A. bovis. However, consensus and concatenated sequence phylogenetic trees showed similar results, revealing 2 subgroups within the genus.

Anaplasma marginale is an etiologic agent of bovine anaplasmosis. This study aimed to molecularly detect and characterize A. marginale that is prevalent in Mongolian cattle populations. A highly specific and sensitive nested PCR (nPCR) method based on the Msp5 gene was developed to detect A. marginale (Msp5 nPCR). The method detected A. marginale from the positive DNA samples obtained from different countries, while no amplicons were observed from DNA samples of several other bovine blood pathogens tested. The detection limit of Msp5 nPCR was determined to be 2 copies/μl. The method was tested against field blood DNA samples prepared from 300 Mongolian cattle in 2010. Results indicated a prevalence rate of 8.7% (26 of 300). On the other hand, partial DNA fragments of an Anaplasma sp. closely related to A. ovis (with 95.0% identity) were detected using a different nPCR method based on groEL gene. The phylogenetic analyses based on the Msp5, groEL and 16S rRNA genes demonstrated that A. marginale isolates in Mongolia were not divergent from the isolates distributed in other countries. The present study successfully established a new nPCR assay that can detect A. marginale, and reported the first molecular detection and characterization of A. marginale and an Anaplasma sp. closely related to A. ovis in Mongolian cattle populations.

Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

Anaplasma marginale has been detected in the Philippines only by peripheral blood smear examination and serological methods. This study generally aimed to molecularly detect and characterize A. marginale in cattle and ticks in Cebu, Philippines. A total of 12 bovine blood samples and 60 Rhipicephalus (Boophilus) microplus ticks were collected on the Cebu Island in 2011. 16S rRNA-based screening-PCR and DNA sequencing revealed 8 cattle (66.7%) and 8 ticks (13.3%) to be positive for A. marginale, and 1 tick (1.7%) to be positive for A. centrale. Selected positive DNA samples were further characterized based on 16S rRNA (longer sequence), Msp5, Msp1α,gltA and groEL genes for phylogenetic analyses. Sequence identities of partial DNA fragments of A. marginale from the Philippines revealed 99.1-100% (16S rRNA, gltA, groEL and Msp5) and 94.3-97.6% (Msp1α) identities to the closest isolates from other countries. Moreover, sequence analysis of the Msp1α, gene showed 3 variants, including a case of co-infection with 2 variants. Phylogenetic analyses based on Msp1α and Msp5 genes revealed that Philippine A. marginale isolates formed a monophyletic lineage, and were phylogenetically related to Brazilian and Chinese isolates. On the other hand, a highly specific and sensitive nested PCR based on groEL, with a detection limit of 2 copies/PCR, was developed to detect A. marginale in the Philippines. This study reported the first molecular detection and characterization of A. marginale in cattle and R. microplus in Cebu, Philippines.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.parint.2016.10.001.The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Babesia vogeli is a tick-borne protozoal pathogen that infects erythrocytes. In Southeast Asia, this pathogen has only been reported in Thailand. In this study, nine dogs presented at three different veterinary clinics in Cebu City, Philippines were found positive for B. vogeli. DNA was extracted from blood samples and tested using a PCR for genus Babesia and a PCR specific for B. vogeli (both based on the 18S rRNA gene). Blood smears (triplicate) from each sample were found negative. All positive amplicons were sequenced and were found to be 99.4% identical to registered B. vogeli sequences at Genbank. Phylogenetic analysis revealed monophyletic grouping of Philippine sequences with the registered A. platys Genbank sequences. This is the first report of B. vogeli infection in dogs in the Philippines.

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.

Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) and tick-borne fever in ruminants. A closely related and potentially novel Anaplasma sp. in Japan was recently characterized. The aims of the study were to provide molecular evidence for the presence of these 2 species in Japan, and to develop a reliable PCR method based on the nucleotide differences within the citrate synthase (gltA) gene. DNA samples from 182 ixodid ticks (134 Ixodes persulcatus, 35 Haemaphysalis douglasii and 13 I. ovatus) collected from 2 sites in Hokkaido, Japan, were screened for A. phagocytophilum and its closely related Anaplasma sp. (herein designated as Anaplasma sp. Japan) using 16S rRNA PCR, revealing a combined prevalence rate of 27.5% (50 samples). The positive samples were then used to evaluate a newly developed gltA-based nested PCR method. Selected positive samples were further characterized using the groEL gene for confirmation and phylogenetic analyses. Two groups of sequence results were obtained: those that had closer identities with (1) A. phagocytophilum (99.5-99.6% for 16S rRNA, 97.5% for gltA and 98.4% for groEL), and those that had closer identities with (2) Anaplasma sp. closely related to A. phagocytophilum in Japan (99.3% for 16S rRNA, 96.4-98.7% for gltA and 97.5-97.9% for groEL). The present study confirmed the distinct presence of A. phagocytophilum and its closely related Anaplasma sp. in Japan, and developed a new PCR detection method based on gltA that can distinguish the 2 organisms.

Background Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya. Methods Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes. Results B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo–derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents. Conclusions The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1106-9) contains supplementary material, which is available to authorized users.

Ehrlichia canis, a canine tick-borne pathogen with wide geographic distribution, has been serologically and molecularly detected in the Philippines. The present study aimed to characterize E. canis detected from Rhipicephalus sanguineus ticks and dogs in Cebu, Philippines, using the RNA polymerase sub-unit Beta (rpoB), a gene that has been used for disease diagnosis and resolution of phylogenetic relationships between closelyrelated species. Using a 16S rRNA gene-based PCR that screens Ehrlichia spp., DNA samples obtained from the blood of 10 dogs, confirmed to be serologically positive for E. canis, were tested and found positive for E. canis after subsequent DNA sequencing. DNA from infected ticks and the 16S rRNA-E. canis-positive canine blood samples from the present study were further analyzed using the rpoB gene. All registered Ehrlichia spp. rpoB gene sequences were aligned to design specific primers that can amplify a partial 1572-bp length sequence of E. canis. The obtained sequences revealed 99.8-100 % identities with each other, and 99.8-100 % and 87.8-89.1 % identities with registered E. canis and E. chaffeensis sequences from the USA, respectively. Phylogenetic analysis revealed that the obtained partial rpOB sequences formed a clade with E. canis strains from the USA. The present study is the first rpoB characterization of E. canis in the Philippines, and apparently in Asia, and provides additional evidence of the presence of the pathogen in the country. It also adds information on the high conservation of the rpoB gene in E. canis., Ehrlichia canis, geografski vrlo proširena rikecija u pasa, dokazana je serološki i molekularno na Filipinima. Određena su obilježja E. canis dokazane u krpelja Rhipicephalus sanguineus i pasa u pokrajini Cebu na Filipinima. To je učinjeno analizom podjedinice β (rpoB) RNA polimeraze, gena koji je rabljen za dijagnosticiranje bolesti i otkrivanje filogenetske srodnosti između usko srodnih vrsta. Upotrebom lančane reakcije polimerazom temeljene na genu 16S rRNA pomoću kojeg se razlikuju vrste roda Ehrlichia, u krvi 10 pasa serološki pozitivnih na E. canis dokazana je DNA specifična za E. canis. DNA iz zaraženih krpelja i uzorci krvi pasa pozitivnih na 16S rRNA-E. canis bili su dalje analizirani na osnovi gena rpoB. Sve dokazane sekvencije gena rpoB rikecija roda Ehrlichia bile su poravnate radi sinteze specifičnih početnica s kojima se može umnožiti slijed specifičan za E. canis dužine 1572 bp. Umnožene sekvencije pokazivale su 99,8-100 % identičnosti međusobno, 99,8-100 % identičnosti s vrstom E. canis iz SAD-a i 87,8-89,1 % identičnosti sa slijedovima E. chaffeensis iz SAD-a. Filogenetska analiza je pokazala da se sekvencije rpoB nalaze u skupini sa sojevima E. canis iz SAD-a. U istraživanju je prvi put analiziran rpoB vrste E. canis na Filipinima i u Aziji. Ono pruža dodatni dokaz prisutnosti te vrste na tom području. Također pruža informaciju o visokoj konzerviranosti gena rpoB vrste E. canis.

Coxiella türleri kene ısırması ile geçebilen patojenik etkendir. Mevcut araştırma Filipinler, Cebu’da Rhipicephalus sanguineus kenelerinden Coxielle türlerinin varlığını moleküler tekniklerle belirlemek için yapıldı. Otuzaltı köpekten elde edilen toplam 164 adet kene Nested Polimeraz Zincir Reaksiyonu (PCR) ile kısmi 16S ribozomal RNA gen bölgesi çoğaltılarak analiz edildi. Beş adet köpekten elde edilen 9 adet kene pozitif (%5.5) bulundu. Sekans analizi sonucunda Coxiella burnetii (%96.3) ve Coxiella sp. endosymbiont (%98.1-100) ile benzer Coxiella spp.'ya ait DNA fragmentleri tespit edildi. Belirlenen DNA fragmentleri birbirleri ile de %98.3-100 oranında benzerdi. Bu çalışma Filipinler Cebu'da Rhipicephalus sanguineus kenelerinden elde edilen Coxielle spp. varlığı- nı gösteren ilk araştırmadır, Coxiella species are known to be potentially pathogenic tickborne organisms. This study was aimed to investigate the presence of Coxiella sp. in Rhipicephalus sanguineus ticks in Cebu, Philippines by molecular based techniques. A total of 164 ticks were collected from 36 dogs and analyzed by a Nested Polymerase Chain Reaction (PCR) for the amplification of partial 16S ribosomal RNA gene region. Nine ticks (5.5%) obtained from 5 dogs were found positive. Sequencing revealed partial DNA fragments of Coxiella spp. which were 96.3% and 98.1-100% identical to Coxiella burnetii and Coxiella sp. endosymbiont, respectively. The detected DNA fragments also shared 98.3-100% identities with each other. This study is the first report on the existence of Coxiella spp. from Rhipicephalus sanguineus ticks in Cebu, Philippines.

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2–100, 73.1–100, and 67.3–100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite.

Vector-borne diseases (VBDs) continue to threaten the worldwide livestock industry, but comprehensive epidemiological surveys on such diseases have not been conducted in the Philippines. In the present study, we screened 408 bovine blood samples from 9 areas in Cebu, Philippines, for various VBD pathogens using specific PCR assays. The results revealed prevalences of 54.7, 15.4, 10.0, and 12.0% for Anaplasma spp., Babesia bigemina, Babesia bovis, and Trypanosoma (Tr.) theileri, respectively. In contrast, none of the samples were positive for Trypanosoma (Tr.) evansi, Theileria (Th.) orientalis, and Theileria (Th.) annulata. Mixed infections were observed in 24.2% of the samples tested. Phylogenetic analysis based on the 16S rRNA gene revealed that the Anaplasma spp. sequences from the present study were genetically close either to Anaplasma marginale or Anaplasma phagocytophilum. In addition, B. bovis RAP-1 and Babesia bigemina AMA-1 gene sequences were identical and monophyletic to other known B. bovis and B. bigemina sequences. On the other hand, Tr. theileri cathepsin-L like protein gene sequences shared 97.1-100% identities with those from the USA and Brazil and clustered within a single genotype in the phylogenetic tree. The molecular identification of several VBD pathogens in Cebu cattle calls for the implementation of control measures to prevent the spread of these pathogens to nearby localities or islands, and ultimately, economic losses to the Philippine economy.

Recent molecular analyses of the Anaplasma sp. closely related to Anaplasma phagocytophilum (previously believed to be A. phagocytophilum) in Japan have clarified its distinct phylogenetic position. PCR methods relying on 16S rRNA- and P44/MSP2-based primers designed to detect this species have low sensitivity and specificity. In this study, a highly sensitive and specific nested PCR method using newly designed primers based on heat-shock operon gene (groEL) was developed to detect this species. The method was later used in an epidemiological study testing DNA samples from 85 Ixodid ticks (collected by flagging) and 50 cattle from the same pastureland in Nakaosobetsu, Hokkaido, Japan. Results revealed prevalence rates of 2.4% (2 of 85) in ticks and 2% (1 of 50) in cattle. The present study also reported the first molecular detection of the Anaplasma sp. closely related to A. phagocytophilum in Japan in H. douglasii, and established a new reliable PCR method that detects this Anaplasma sp. closely related to A. phagocytophilum in Japan.

Ehrlichia canis infection of dogs in the Philippines has been detected by serological and peripheral blood smear examination methods, but not by molecular means. Anaplasma platys infection in dogs has not yet been officially reported, although it is suspected to occur in the country. Thus, sensitive and specific molecular techniques were used in this study to demonstrate the presence of both E. canis and A. platys in the Philippines. A total of 164 Rhipicephalus sanguineus ticks was collected from 36 dogs. Seven tick samples were found positive with E. canis and one sample with A. platys. To further characterize these pathogens, molecular analyses based on citrate synthase and heat-shock operon genes were also performed. Philippine strains were found to be not divergent from strains from other countries. The present results are the first molecular detection and analyses of E. canis and A. platys in ticks from dogs in the Philippines.

Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

Theileria orientalis and hemotropic Mycoplasma spp. (hemoplasmas) can cause anemia in cattle. Cattle infected with one of these two pathogens tend to resist infection by the other pathogen. This is called the “interference phenomenon”. However, the detailed investigation of this phenomenon using molecular techniques has not been performed until now. We used PCR to analyze blood samples from cattle grazing in two pastures, and investigated interference between T. orientalis and hemoplasmas. Results indicate that single infection with T. orientalis or hemoplasmas was more common than co-infections. The degree of anemia observed in co-infected animals was significantly milder compared to those only infected with T. orientalis. These findings revealed that the interaction between these two pathogens in cattle demonstrated interference.

The prevalence and risk factors of 2 bovine hemoplasma species-Mycoplasma wenyonii (Mw) and "Candidatus Mycoplasma haemobos" (CMh)-were examined by direct PCR using whole blood samples collected from 343 cattle in Eastern Hokkaido, Japan. The sensitivity of the direct PCR assay was 10-fold higher than the standard PCR assay, detecting 5 copies of bovine hemoplasma DNA per reaction. An epidemiological survey of sampling location, living conditions, age and bovine leukemia virus antibody was performed to assess infection risk factors. The prevalence rates were 38.5% for Mw and 39.1% for CMh, respectively, with an overall prevalence rate of 64.7% for all bovine hemoplasma infections. Significant differences were found in sampling location, living conditions and age. Compared with pastured cattle, farmed cattle were significantly more susceptible to infection with total hemoplasma species (P