Heartworm adulticide treatment: a tropical perspective.

Dirofilaria immitis (the canine heartworm) is widespread in the tropics, with prevalence surpassing 30% in high-risk areas. In addition to the suitable climatic conditions that favour mosquito abundance and filarial larva development, there is low compliance with the recommended year-round use of preventives in these transmission hotspots. This represents a major concern, considering that melarsomine (first-line heartworm adulticide) is unavailable in several tropical countries, resulting in the so-called slow-kill protocol being the only available adulticide treatment option. In this article, the members of TroCCAP (Tropical Council for Companion Animal Parasites) review the current distribution of heartworm in the tropics and the availability of melarsomine, and discuss alternatives for the management of heartworm infections in dogs.

TroCCAP recommendations for the diagnosis, prevention and treatment of parasitic infections in dogs and cats in the tropics.

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.

Anaplasma species of veterinary importance in Japan.

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.

Multiple infections of Anaplasma platys variants in Philippine dogs.

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.

Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya.

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.

High genetic diversity of Anaplasma marginale detected from Philippine cattle.

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 phylogenetic position of Anaplasma bovis and inferences on the phylogeny of the genus Anaplasma.

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.

Diversity of Babesia bovis merozoite surface antigen genes in the 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.

Molecular survey of bovine vector-borne pathogens in Cebu, Philippines.

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.

Survey on tick-borne pathogens in thoroughbred horses in the Hidaka district, Hokkaido, Japan.

A total of 87 Thoroughbred horses and 10 ixodid ticks from a ranch in Hidaka district, Hokkaido were tested for tick-borne diseases. Using the indirect fluorescent antibody (IFA) method, 3.4, 92.0 and 97.7% of the horses showed antibody titers of ≥ 80 against Anaplasma phagocytophilum, Rickettsia helvetica, and Borrelia garinii, respectively. This is the first report of infection with the 3 pathogens in horses in Japan. Using PCR, DNAs from the peripheral blood of all horses were found negative with any Anaplasma, Rickettsia and Borrelia spp., while those from Haemaphysalis megaspinosa ticks were found positive for Anaplasma sp. closely related to A. phagocytophilum in Japan, and A. bovis. B. japonica was also detected in an H. flava tick for the first time.

First molecular characterization of Anaplasma marginale in cattle and Rhipicephalus (Boophilus) microplus ticks in Cebu, Philippines.

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.

Specific molecular detection and characterization of Anaplasma marginale in Mongolian cattle.

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.

Dual presence of Anaplasma phagocytophilum and its closely related Anaplasma sp. in ixodid ticks in Hokkaido, Japan, and their specific molecular detection.

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.