A Survey of Canine Heartworm Awareness in Grenada,West Indies

Heartworm is a common parasite of dogs in Grenada, West Indies, due to the tropical climate and large number of mosquitoes. Because Grenada is a developing country and a small island, resources and education on heartworm are limited. In an effort to raise awareness of canine heartworm and the preventive measures available, observations and surveys were performed to determine the following: (a) heartworm knowledge among Grenadian dog caregivers seeking veterinary care at the Grenada Society for the Prevention of Cruelty to Animals (GSPCA), (b) dissemination of heartworm information by GSPCA staff to Grenadian dog caregivers, and (c) reasons Grenadians are not administering heartworm prevention to their dogs. Results indicated a marked deficiency in both heartworm knowledge among Grenadian dog caregivers and heartworm knowledge acquisition and dissemination. Lack of knowledge was also cited as a major reason Grenadian dog caregivers were not using heartworm prevention. These results are currently being utilized to modify heartworm education practices at the GSPCA by incorporating Grenada-specific educational tools, with the ultimate goal of reducing illness and death by canine heartworm.     

An MDR1 promoter allele with higher promoter activity is common in clinically isolated strains of Candida albicans

In the opportunistic fungal pathogen Candida albicans, up-regulation of MDR1, encoding an efflux transporter, leads to increased resistance to the antifungal drug fluconazole. Antifungal resistance has been linked to several types of genetic change in C. albicans, including changes in genome structure, genetic alteration of the drug target, and overexpression of transporters. High-level over-expression of MDR1 is commonly mediated by mutation in a trans-acting factor, Mrr1p. This report describes a second mechanism that contributes to up-regulation of MDR1 expression. By analyzing the sequence of the MDR1 promoter region in fluconazole-resistant and fluconazole-susceptible strains, we identified sequence polymorphisms that defined two linkage groups, corresponding to the two alleles in the diploid genome. One of the alleles conferred higher MDR1 expression compared with the other allele. Strains in which both alleles were of the higher activity type were common in collections of clinically isolated strains while strains carrying only the less active allele were rare. As increased expression of MDR1 confers higher resistance to drugs, strains with the more active MDR1 promoter allele may grow or survive longer when exposed to drugs or other selective pressures, providing greater opportunity for mutations that confer high-level drug resistance to arise. Through this mechanism, higher activity alleles of the MDR1 promoter could promote the development of drug resistance.     

Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism

Zoonotic visceral leishmaniasis (VL) is a disease of dogs, humans and other animals caused by the intracellular macrophage parasite Leishmania infantum. We examined the relationship between DLA class II alleles ( DRB1, DQA1, DQB1) and the course of infection in a cohort of Brazilian mongrel dogs exposed to natural L. infantum infection. DLA alleles were typed by sequence-based typing. DLA-DRB1 genotype was significantly associated with levels of anti- Leishmania IgG and parasite status assessed by PCR. Dogs with DLA-DRB1*01502 had higher levels of specific IgG and an increased risk of being parasite positive compared with dogs without this allele, controlling for other alleles and significant variables. No significant associations were seen for DLA-DQA1 or DLA-DQB1 alleles. These results suggest that the DLA-DRB1 locus plays a role in determining susceptibility to canine VL. As the domestic dog is the main reservoir for human infection, the identification of genetic factors influencing canine resistance or susceptibility to VL may provide insights into the immunology and potential control through vaccination of VL.     

Modeling the distribution and abundance of the non-native parasite, canine heartworm, in California coyotes

We applied a habitat-modeling approach using logistic regression to predict the distribution and abundance of canine heartworm ( Dirofilaria immitis ) in coyotes ( Canis latrans ) throughout California. Heartworm is an arthropod-borne parasite of considerable economic and ecological importance. In California, coyotes serve as the primary sylvatic maintenance host and represent a useful sentinel for this parasite. To develop the model, we used a large collection of coyote blood specimens and carcasses collected from spatially broad, yet nonrandom, locations in California. Survey data were useful in refining previous coarser models that predicted uniformly high prevalence of heartworm throughout the coastal and Sierra-Nevada foothills, by indicating variability within this broadly defined plant-climate zone. Due to the non-random nature and large spatial scale of our data-set, we restricted variables to those thought to be most generally important. Modeling indicated that woodlands with a relatively dense canopy, suitable breeding and host-seeking habitat of the western treehole mosquito ( Ochlerotatus sierrensis ), were a good predictor of heartworm prevalence. Within this habitat, prevalence increased with precipitation, which likely affected mosquito abundance. The distribution of heartworm was limited to areas with average cumulative temperatures high enough to enable larval development of heartworms within their mosquito vectors. The prediction accuracy of our model was supported by goodness-of-fit tests, cross-validation tests and external validation tests. The model provided a useful guide to the relative risk of heartworm exposure in California, although the resolution was necessarily coarse and prevalence estimates related to risk in an ordinal manner only.     

Drug resistance in diploid yeast is acquired through dominant alleles, haploinsufficiency,gene duplication and aneuploidy

Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.     

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.     

Diseases, parasites and survival of coyotes in south-central Georgia.

Serologic testing, radio-telemetry and post-mortem diagnostic evaluations were used to investigate survival and causes of mortality among 17 coyotes (Canis latrans) in south-central Georgia (USA). Prevalence of canine heartworm (Dirofilaria immitis) microfilariae was lower (P = 0.057) among fall-captured (22%) than among winter-captured (75%) coyotes. Prevalence of heartworm was higher among adults than juveniles in the fall, but no significant difference was detected between animals captured in winter. Antibodies were found against canine parvovirus (65%), canine parainfluenza virus (59%), infectious canine hepatitis virus (41%), and Toxoplasma gondii (18%). Antibodies were not found to Brucella canis, canine coronavirus, five serovars of Leptospira interrogans, or canine distemper virus. Seroprevalence of canine parvovirus was lower (P = 0.009) among fall-captured animals (33%) than winter-captured animals (100%). The Kaplan-Meier estimate of annual survival was 0.500 for all animals. Juvenile survival did not differ (P = 0.79) from adult survival, but male survival (S = 0.217) was lower (P = 0.11) than female survival (S = 0.804). Two of nine (22%) mortalities were human-caused, one was due to concurrent canine parvovirus and canine distemper virus infections, one animal died of trauma, two were considered natural mortalities of unknown cause, and no cause of death could be determined for the remaining three animals. Natural mortality may be significant for coyotes in south-central Georgia, although there was no apparent link between exposure to pathogens and the animals' subsequent fate in our small sample.     

Prevalence of dirofilarial infection in raccoon dogs in Japan.

The raccoon dog (Nyctereutes procyonoides viverrinus) is known to acquire canine heartworm (Dirofilaria immitis) infection. We surveyed the prevalence of heartworm infection in free-ranging raccoon dogs in the Nishi-Tama (Tokyo) and Kanagawa areas of Japan. A total of 75 raccoon dog carcasses, including 29 animals from the Nishi-Tama area and 46 from the Kanagawa area, were necropsied between 1992 and 1993. Eight out of 75 raccoon dogs were found to be infected (overall 10.7%). The prevalence of infection was 6 and 16% in Nishi-Tama and Kanagawa, respectively. Microfilarial production was observed in the uterus of one female adult dog.     

Angiostrongylus vasorum: a real heartbreaker

Recent reports suggest that the canine heartworm Angiostrongylus vasorum is expanding from traditional endemic foci in several parts of the world. We are ill placed to judge the causes and potential consequences of this expansion because of a lack of knowledge about fundamental aspects of the biology of the parasite. We call for a renewed focus on this important but neglected nematode.     

Morbidity-mortality factors and survival of an urban coyote population in Arizona

The health of coyotes (Canis latrans) in urban areas has not been studied. Our objectives were to assess the health of coyotes in Tucson (Arizona, USA) by determining the prevalence of antibodies to selected pathogens, estimating survival rates, and identifying sources of mortality. We drew blood from 22 coyotes to evaluate the prevalence of heartworm (Dirofilaria immitis) antigens, and antibodies to canine distemper virus (CDV), infectious canine hepatitis (ICH), canine parvovirus (CPV), and seven serovars of Leptospira interrogans. We trapped and radiocollared 19 coyotes to determine survival rates. We performed necropsies on 19 coyotes to quantify their general health, the presence of internal and external parasites, and causes of mortality. No coyotes tested positive for heartworm antigens. The prevalence of antibody to CDV, ICH, and CPV was 27, 50, and 100%, respectively. Twenty-seven percent of coyotes tested positive for one of five serovars of L. interrogans. The diseases for which coyotes in Tucson possessed antibodies appear to be enzootic in the population. The annual survival rate of coyotes was 0.72. Eleven necropsied coyotes were killed by cars, five coyotes were hit by cars, two were killed by a trapper, and the cause of death for one coyote was unknown. Coyotes in Tucson appear to be exposed to the viral, bacterial, and parasitic infections common in many coyote populations, but humans are the major source of mortality.     

Expansion of the canine A blood group system

A detailed study of the canine A blood group system was undertaken, resulting in the expansion of this system into a three-factor, four-allelic one with the recognition of an additional subtype, a3. The serological and extensive family data supported the proposed genetic theory of four alleles with dominance with the order being Aa1, Aa2, Aa3 and A-. Gene frequencies of the alleles were determined in various breeds of dogs with frequencies in the general Brisbane population being 0.244 (Aa1), 0.042 (Aa2), 0.045 (Aa3) and 0.669 (A-).     

Spatial and temporal transmission risk of Dirofilaria immitis in Argentina

The aim of this work was to assess spatial and seasonal Dirofilaria immitis transmission risk throughout Argentina with models based on the temperature threshold below which filarial development will not proceed in the mosquito (i.e. 14 °C), the occurrence and the number of potential vector mosquito species, and the Heartworm Development Units derived from the degree-days concept. The four models showed a similar increasing southwest-northeast tendency and correlated significantly with canine prevalences used as external validation data. About one-third of Argentina would be suitable for heartworm transmission and the highest risk areas include the north-eastern provinces. According to our models, heartworm transmission is markedly seasonal with peaks in January and February; no region would support transmission throughout the year. To improve the present models, it is necessary to know which mosquito species are competent rather than potential vectors in the country. We believe the present study provides the first risk assessment maps for D. immitis transmission in the Southern Hemisphere and provides a useful guide for heartworm prevention during the transmission periods in different regions of Argentina.     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding β-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two β-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.     

Efficacy of moxidectin for the prevention of adult heartworm (Dirofilaria immitis) infection in dogs

The authors report the efficacy of orally administered moxidectin for the prevention of canine heartworm infection in two endemic areas in northern Italy. Two trials were conducted on a total of 257 dogs, including 137 treated with moxidectin (minimum dose of 3 mcg/kg body weight), 85 with ivermectin (minimum dose 6.6 mcg/kg b.w.) and 35 untreated controls. Results of testing for microfilariae and circulating adult female antigens were negative for all treated dogs at the end of both trials. No adverse reactions to moxidectin were observed. In the study areas, prevalence values for Dirofilaria immitis infection calculated on the basis of the untreated controls and testing dogs which had no preventive treatment in the previous transmission season ranged 23-65%. This study confirms the efficacy and safety of moxidectin in the prevention of adult heartworm infection in dogs.     

Reverse Chemical Genetics: Comprehensive Fitness Profiling Reveals the Spectrum of Drug Target Interactions

The emergence and prevalence of drug resistance demands streamlined strategies to identify drug resistant variants in a fast, systematic and cost-effective way. Methods commonly used to understand and predict drug resistance rely on limited clinical studies from patients who are refractory to drugs or on laborious evolution experiments with poor coverage of the gene variants. Here, we report an integrative functional variomics methodology combining deep sequencing and a Bayesian statistical model to provide a comprehensive list of drug resistance alleles from complex variant populations. Dihydrofolate reductase, the target of methotrexate chemotherapy drug, was used as a model to identify functional mutant alleles correlated with methotrexate resistance. This systematic approach identified previously reported resistance mutations, as well as novel point mutations that were validated in vivo. Use of this systematic strategy as a routine diagnostics tool widens the scope of successful drug research and development.     

Isolations of Cry2Ab resistance in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae) are allelic

Alleles conferring resistance to Cry2Ab toxin occur at a frequency of 0.0033 in Australian populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), and it is evident that detectable levels of resistance predated the introduction of transgenic cotton expressing this toxin. From 2002 until 2006, 10 such resistant alleles were scored. Here, we examine colonies established from five of the 10 isolates by using complementation tests to determine their genetic relationships. The results demonstrate that the resistance in each colony is due to alleles at the same locus and that for each allele the resistance is recessive. This latter finding is in conflict with the frequency of apparently resistant individuals occurring in the initial F2 tests that were used to identify alleles that confer resistance. These frequencies were variable (range 6.7-35.6%, mean 16.2%), but they generally indicated a measure of dominance (i.e., were >6.25% expected for recessive resistance). We hypothesize that this conflict is the result of differences in the genetic background of the laboratory adapted resistant colonies and the initial field isolations.     

Non-linear selection response in Drosophila: a strategy for testing the rare-alleles model of quantitative genetic variability

Quantitative genetic theory predicts that variation due to rare alleles at many loci will generate a transient acceleration in the response to directional selection. We have tested this prediction by constructing experimental lines ofDrosophila melanogaster that carry positively selected ethanol resistance alleles at low frequencies, and then subjecting the lines to directional selection for ethanol resistance. Approximately 468,000 files were subjected to artificial selection over 30 generations. The predicted non-linear selection responses were observed in all experimental lines and replicates, on three genetic backgrounds. In contrast, un-selected controls and lines carrying random alleles at low frequencies on the same genetic backgrounds exhibited linear selection responses. These results demonstrate that non-linearities due to rare alleles are detectable and repeatable, provided that experiments are done on a sufficiently large scale. The results suggest that it may be possible to test for rare-alleles as a component of naturally occurring genetic variation by careful examination of selection response curves.     

A random model for mapping imprinted quantitative trait loci in a structured pedigree: an implication for mapping canine hip dysplasia

Genetic imprinting may have played a more notable role in shaping embryonic development of plants, animals, and humans than previously appreciated. Quantitative trait loci that are imprinted (iQTL) exert monoallelic effects, depending on the parent of origin, which is an exception to the laws of Mendelian genetics. In this article, we present a modified random effect-based mapping model to use in a genome-wide scan for the distribution of iQTL that contribute to genetic variance for a complex trait in a structured pedigree. This model, implemented with the maximum likelihood method, capitalizes on a network of relatedness for maternally and paternally derived alleles through identical-by-descent sharing, thus allowing for the discrimination of the genetic variances due to alleles derived from maternal and paternal parents. The model was employed to map iQTL responsible for canine hip dysplasia in a multihierarchical canine pedigree, founded with seven greyhounds and six Labrador retrievers. Of eight significant QTL detected, three, located on CFA1, CFA8, and CF28, were found to trigger significant parent-of-origin effects on the age of femoral capital ossification measured at the left and right hips of a canine. The detected iQTL provide important candidate regions for fine-mapping of imprinted genes and for studying their structure and function in the control of complex traits.     

Genetic Basis of Haloperidol Resistance in Saccharomyces cerevisiae Is Complex and Dose Dependent

The genetic basis of most heritable traits is complex. Inhibitory compounds and their effects in model organisms have been used in many studies to gain insights into the genetic architecture underlying quantitative traits. However, the differential effect of compound concentration has not been studied in detail. In this study, we used a large segregant panel from a cross between two genetically divergent yeast strains, BY4724 (a laboratory strain) and RM11_1a (a vineyard strain), to study the genetic basis of variation in response to different doses of a drug. Linkage analysis revealed that the genetic architecture of resistance to the small-molecule therapeutic drug haloperidol is highly dose-dependent. Some of the loci identified had effects only at low doses of haloperidol, while other loci had effects primarily at higher concentrations of the drug. We show that a major QTL affecting resistance across all concentrations of haloperidol is caused by polymorphisms in SWH1, a homologue of human oxysterol binding protein. We identify a complex set of interactions among the alleles of the genes SWH1, MKT1, and IRA2 that are most pronounced at a haloperidol dose of 200 µM and are only observed when the remainder of the genome is of the RM background. Our results provide further insight into the genetic basis of drug resistance.     

Dirofilaria immitis in the dingo (Canis familiaris dingo) in a tropical region of the Northern Territory, Australia

The heart and lungs from 32 adult dingoes (Canis familiaris dingo) were examined for canine heartworm (Dirofilaria immitis) infection. Eighteen of 32 (56%) samples were infected, with intensity of infection ranging from 1 to 31 worms per animal. Seven of 18 (39%) infections were single sex infections. Large numbers of circulating microfilariae were present in blood from all dingoes infected with both sexes of worms.