Rabies prevention and management of cats in the context of trap-neuter-vaccinate-release programmes.

Domestic cats are an important part of many Americans' lives, but effective control of the 60-100 million feral cats living throughout the country remains problematic. Although trap-neuter-vaccinate-return (TNVR) programmes are growing in popularity as alternatives to euthanizing feral cats, their ability to adequately address disease threats and population growth within managed cat colonies is dubious. Rabies transmission via feral cats is a particular concern as demonstrated by the significant proportion of rabies post-exposure prophylaxis associated with exposures involving cats. Moreover, TNVR has not been shown to reliably reduce feral cat colony populations because of low implementation rates, inconsistent maintenance and immigration of unsterilized cats into colonies. For these reasons, TNVR programmes are not effective methods for reducing public health concerns or for controlling feral cat populations. Instead, responsible pet ownership, universal rabies vaccination of pets and removal of strays remain integral components to control rabies and other diseases.

A conceptual model for the impact of climate change on fox rabies in Alaska, 1980-2010.

The direct and interactive effects of climate change on host species and infectious disease dynamics are likely to initially manifest\ at latitudinal extremes. As such, Alaska represents a region in the United States for introspection on climate change and disease. Rabies is enzootic among arctic foxes (Vulpes lagopus) throughout the northern polar region. In Alaska, arctic and red foxes (Vulpes vulpes) are reservoirs for rabies, with most domestic animal and wildlife cases reported from northern and western coastal Alaska. Based on passive surveillance, a pronounced seasonal trend in rabid foxes occurs in Alaska, with a peak in winter and spring. This study describes climatic factors that may be associated with reported cyclic rabies occurrence. Based upon probabilistic modelling, a stronger seasonal effect in reported fox rabies cases appears at higher latitudes in Alaska, and rabies in arctic foxes appear disproportionately affected by climatic factors in comparison with red foxes. As temperatures continue a warming trend, a decrease in reported rabid arctic foxes may be expected. The overall epidemiology of rabies in Alaska is likely to shift to increased viral transmission among red foxes as the primary reservoir in the region. Information on fox and lemming demographics, in addition to enhanced rabies surveillance among foxes at finer geographic scales, will be critical to develop more comprehensive models for rabies virus transmission in the region.

Bait acceptability for delivery of oral rabies vaccine to free-ranging dogs on the Navajo and Hopi Nations.

In many areas of the world, only 30 to 50% of dogs are vaccinated against rabies. On some US Indian Reservations, vaccination rates may be as low as 5 to 20%. In 2003 and 2004, we evaluated the effectiveness of commercially available baits to deliver oral rabies vaccine to feral and free-ranging dogs on the Navajo and Hopi Nations. Dogs were offered one of the following baits containing a plastic packet filled with placebo vaccine: vegetable shortening-based Ontario slim baits (Artemis Technologies, Inc.), fish-meal-crumble coated sachets (Merial, Ltd.), dog food polymer baits (Bait-Tek, Inc.), or fish meal polymer baits (Bait-Tek, Inc.). One bait was offered to each animal and its behaviour toward the bait was recorded. Behaviours included: bait ignored, bait swallowed whole, bait chewed and discarded (sachet intact), bait chewed and discarded (sachet punctured), or bait chewed and consumed (sachet punctured). Bait acceptance ranged from 30.7% to 77.8% with the fish-meal-crumble coated sachets having the highest acceptance rate of the tested baits.

Downside risk of wildlife translocation.

Translocation has been used successfully by wildlife professionals to enhance or reintroduce populations of rare or extirpated wildlife, provide hunting or wildlife viewing opportunities, farm wild game, and reduce local human-wildlife conflicts. However, accidental and intentional translocations may have multiple unintended negative consequences, including increased stress and mortality of relocated animals, negative impacts on resident animals at release sites, increased conflicts with human interests, and the spread of diseases. Many wildlife professionals now question the practice of translocation, particularly in light of the need to contain or eliminate high profile, economically important wildlife diseases and because using this technique may jeopardize international wildlife disease management initiatives to control rabies in raccoons, coyotes, and foxes in North America. Incidents have been documented where specific rabies variants (Texas gray fox, canine variant in coyotes, and raccoon) have been moved well beyond their current range as a result of translocation, including the emergence of raccoon rabies in the eastern United States. Here, we review and discuss the substantial challenges of curtailing translocation in the USA, focusing on movement of animals by the public, nuisance wildlife control operators, and wildlife rehabilitators.

Attaining raccoon rabies management goals: history and challenges.

Prior to 1977, raccoon (Procyon lotor) rabies was confined to the southeastern US. Translocations led to emergence of this rabies variant in the mid-Atlantic states, followed by spread northerly to northeast Ohio and Ontario, Quebec, and New Brunswick, Canada. Raccoon rabies is currently contiguous from southwest Alabama to southeastern Canada. Since 1998, state, federal, county and municipal as well as Canadian and Mexican experts have collaborated on goals and strategies to prevent raccoon rabies spread in North America. Coordinated programmes have been established from Maine to Alabama. Successes have been realized through strategies that rely heavily on oral vaccination. International coordination targeting raccoon rabies continues in eastern Canada, where contingency actions have led to elimination or near elimination in Ontario and New Brunswick. However, increasingly, focus in the US has been directed toward contingency actions to "hold-the-line" where raccoon rabies threatens to spread to new areas, rather than on raccoon rabies elimination. We report on the challenges of achieving enhanced rabies surveillance, containment of raccoon rabies, and local elimination of raccoon rabies, as well as the need for international coordination in meeting these challenges.

Vaccination in conservation medicine.

Unprecedented human population growth and anthropogenic environmental changes have resulted in increased numbers of people living in closer contact with more animals (wild, domestic, and peridomestic) than at any other time in history. Intimate linkage of human and animal health is not a new phenomenon. However, the global scope of contemporary zoonoses has no historical precedent. Indeed, most human infectious diseases classed as emerging are zoonotic, and many of these have spilled over from natural wildlife reservoirs into humans either directly or via domestic or peridomestic animals. Conservation medicine has recently emerged as a meaningful discipline to address the intersection of animal, human, and ecosystem health. Interest in the development of novel vaccines for wildlife encounters important challenges that may prevent progress beyond the conceptual phase. Although notable examples of successful wildlife immunisation programmes exist, depending upon key considerations, vaccination may or may not prove to be effective in the field. When implemented, wildlife vaccination requires a combination of novel zoonosis pathogen management strategies and public education to balance conservation, economic, and public health issues.

Control and prevention of rabies in animals: paradigm shifts.

Animal management is the keystone of any modern programme for the prevention and control of rabies. Historically, "animal control" for local elimination of disease was largely equated with population reduction. However, with relatively few exceptions, culling alone has not led to effective control of rabies. In most documented examples of effective control of rabies in the 20th century, an integrated management approach was used that included public education, responsible stewardship of animal populations, manipulation of the population carrying capacity of the local habitat, and vaccination strategies. Globally, the greatest burden on human health that is attributable to this zoonosis is caused by uncontrolled rabies in dogs. Where political willingness, biomedical infrastructure, and economic stability permit the sustained use of control measures (e.g. stray animal removal and mandatory parenteral vaccination), canine rabies has been significantly suppressed and even eliminated over large geographical areas. Examples include many island nations, most of North America, Europe, and increasingly in South America. Despite the effectiveness of such proven control techniques, however, their implementation in parts of Asia, Africa, and elsewhere has been limited, primarily because of a lack of dedicated resources and intersectoral cooperation, and also because of the burden of high-density populations of dogs. Implementation is often complicated by cultural and social factors, e.g. reluctance to cull apparently ownerless, nuisance animals that are suspected to have been exposed to rabies, partly on the basis of religious beliefs). Attempts to modify animal fertility (such as the encouragement of voluntary spay-neuter programmes or individual chemical contraception, and the extension of such actions to animals in the community) may provide ancillary support in line with other traditional methods of control of canine rabies. With the identification of complex situations in which wildlife rabies persists despite the elimination of canine rabies, e.g. in North America and Europe, cats can pose a significant public health risk requiring consideration of alternative approaches. In any model system, the threat of translocation of infected animals, unintentional or otherwise, provides a strong rationale for the creation of barriers to prevent reintroduction or exacerbation of the disease, and the maintenance of a minimum body of expertise related to surveillance, diagnosis, and the enactment of mitigating measures. While control activities have traditionally focused upon certain Carnivora species, bats represent another worldwide rabies reservoir. Indiscriminate killing of bats and destruction of roosts was once the norm, but such activities are not sanctioned by reputable organizations today. Even vampire bats, responsible for substantial effects on health and agricultural losses in the New World (Mexico to Argentina), should be targeted only by specific control applications, rather than by more widespread, unconventional, non-specific methodology. Bats should be excluded from human living quarters. Implementing measures to prevent bats from gaining access to homes should occur at an appropriate time when the bats are absent, especially to avoid sealing the non-flying young within a building. Although great progress has been made during the past four decades in the induction of herd immunity among free-ranging carnivores via oral vaccination against rabies, similar novel solutions have not been readily applied to bat populations. Given these challenges, new paradigm shifts are eagerly anticipated as additional biotechnological applications (including contraceptives and anticoagulants) are developed to deal with domestic animals and wildlife.

Evaluation and significance of tetracycline stability in rabies vaccine baits.

Tetracycline is widely used as a biomarker for bait consumption by wildlife; tetracycline is incorporated into bones and teeth and can be detected by fluorescence microscopy several weeks postconsumption. During 2003, the United States Department of Agriculture distributed more than 10 million tetracycline-containing rabies-vaccine baits to control the spread of wildlife vectored rabies to humans, pets, and livestock. To estimate the percentage of target species consuming the baits, raccoons and skunks were collected in baited areas and teeth were analyzed for the presence of the biomarker. Several incidents of low biomarker detection rates prompted an investigation of the stability of the biomarker in the baits. Baits were collected at several points along the manufacturing and distribution chain. Baits were analyzed for free and polymer-bound tetracycline and the less active isomer epitetracycline. Results indicated that a portion of the tetracycline was converted to epitetracycline. Additionally, significant quantities of both compounds were trapped in the polymer, which is homogeneously distributed throughout the bait. The results of this study suggest that approximately 40% of the target quantity of tetracycline was unavailable for absorption. This situation could contribute to low biomarker detection rates and suggests that formulation modification should be considered.

Oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control.

Rabies is an acute, progressive, fatal encephalitis caused by viruses in the Family Rhabdoviridae, Genus Lyssavirus. Rabies virus is the representative member of the group. Warm-blooded vertebrates are susceptible to experimental infection, but major primary hosts for disease perpetuation encompass bats and mammalian carnivores. The dog is the global reservoir, and important wild carnivores include foxes, raccoons, skunks, and mongoose, among others. Traditionally, reliance upon long-term, widespread, government-supported programmes aimed at population reduction of animals at risk has been unsuccessful as the sole means of rabies control, based in part upon economical, ecological and ethical grounds. In contrast, immunization of domestic dogs with traditional veterinary vaccines by the parenteral route led to the virtual extinction of canine-transmitted rabies in developed countries. Taken from this basic concept of applied herd immunity, the idea of wildlife vaccination was conceived during the 1960s, and modified-live rabies viruses were used for the experimental oral vaccination of carnivores by the 1970s. The development of safe and effective rabies virus vaccines applied in attractive baits resulted in the first field trials in Switzerland in 1978. Thereafter, technical improvements occurred in vaccine quality and production, including the design of recombinant viruses, as well as in the ease of mass distribution of millions of edible baits over large geographical areas. Over the past few decades, extensive oral vaccination programmes focusing upon the red fox, using hand and aerial distribution of vaccine-laden baits, have resulted in the virtual disappearance of rabies in Western Europe. The same dramatic observation held true for southern Ontario. During the 1990s in the United States, oral vaccination programmes concentrated upon raccoons, grey foxes, and coyotes, with similar success. For example, raccoon rabies has not spread west of the current focus in the eastern states, grey fox rabies is contained in west central Texas, and no recent cases of rabies have been reported from coyotes away from the Mexican border for several years. Despite the progress observed and the absence of substantive adverse environmental or health effects, oral vaccination is not a panacea, and should be viewed as an important adjunct to traditional prevention and control techniques in human and veterinary medicine. Local outbreak suppression of rabies among free-ranging wildlife is documented, and regional elimination of particular virus variants among specific, targeted carnivore hosts is demonstrable, but true disease eradication is not achievable at the present time by current techniques. For example, no practical vaccination methods have been designed for bats. Although lyssaviruses appear in relative compartmentalization between the Chiroptera and Carnivora, major spillover events have been detected from bats to carnivores, and phylogenetic analyses suggest a historical basis for extant viral origins due to interactions between these taxa. Thus, bio-political considerations aside, the possibility for pathogen emergence resulting from transmission by rabid bats with subsequent perpetuation among other animals cannot be discounted easily on any continent, with the possible exception of Antarctica. Clearly, given their biodiversity, distribution, and abundance, novel methods would be necessary to consider meaningful control of rabies in these unique volant mammals. Newer approaches in biotechnology may be envisaged some day for eventual extension to bats, as well as more widespread application to global canine rabies remediation in developing countries.

RS-33295-198: a novel, potent modulator of P-glycoprotein-mediated multidrug resistance.

A novel multidrug resistance modulator, RS-33295-198, circumvented drug resistance in human, mouse, and Chinese hamster cell lines overexpressing P-glycoprotein. It enhanced the antiproliferative activity of doxorubicin, vincristine, etoposide, and paclitaxel and increased doxorubicin retention in multidrug-resistant hamster CHRC5 cells. RS-33295-198 modulated doxorubicin resistance in a murine P388/ADR leukemia model when administered ip via continuous minipump delivery, ip by bolus injection, and orally; it also improved the efficacy of vincristine toward P388/VCR leukemia when given ip or po. RS-33295-198 showed weak activity in enhancing doxorubicin efficacy against a multidrug-resistant human sarcoma xenograft.

Inhibitors of protein tyrosine kinase pp60v-src: sterol sulfates from the brittle star Ophiarachna incrassata.

Bioactivity-guided fractionation of the extracts of the green brittle star Ophiarachna incrassata using a protein tyrosine kinase pp60v-src inhibition assay led to the isolation of a new sterol sulfate [2] together with four known ones (1,3-5). All five compounds were found to inhibit protein tyrosine kinase pp 60v-src.

The marine natural product, halistanol trisulfate, inhibits pp60v-src protein tyrosine kinase activity.

Halistanol trisulfate, a sulfated steroid derivative, was isolated from the extracts of two different marine sponges (genus Topsentia) by bioassay-guided fractionation. It exhibited an IC50 of approximately 4 microM against pp60v-src, the oncogenic protein tyrosine kinase encoded by Rous sarcoma virus. Removing the sulfate groups by acid hydrolysis produced the inactive tris-alcohol, halistanol. The kinetics of inhibition were examined and revealed that halistanol trisulfate is a competitive inhibitor with respect to the peptide substrate, [val5]-angiotensin II, and a mixed inhibitor with respect to ATP. A number of monosulfated steroids were studied for protein tyrosine kinase inhibitory activity, but were found to be inactive.

Anti-tumor activity of mycophenolate mofetil against human and mouse tumors in vivo.

Cultured tumor cell lines, tumor xenografts grown in athymic nude mice, and a murine experimental metastasis model were used to assess the in vitro and in vivo anti-tumor activity of the potent IMP dehydrogenase (IMPDH) inhibitor, mycophenolic acid (MPA), and its morpholinoethyl ester pro-drug, mycophenolate mofetil (MM). The growth of all the cell lines tested was inhibited by MPA in vitro, with EC50 values ranging from less than 0.1 microM to 3.9 microM. Mice were monitored for s.c. tumor outgrowth in the case of human tumor xenograft models or survival time for the murine experimental metastasis model. Treatment with MM p.o. was started 24 hr after tumor challenge or after tumors became palpable. Treatment of athymic nude mice bearing A3.01 (T-lymphoblast), Molt-4 (T-cell leukemia), CaPan-2 (pancreatic adenocarcinoma), CaLu-3 (non-small-cell lung adenocarcinoma), LS174T and T84 (colon adenocarcinoma), and Daudi (B-cell lymphoma) human tumor xenografts with MM significantly inhibited s.c. tumor growth. Treatment of BALB/c mice with MM after i.v. injection of murine RAW117-H10 lymphoma cells in an experimental metastasis assay resulted in increased survival time for treated animals. No significant inhibitory effect on s.c. tumor outgrowth was seen with MM treatment of SK-Hep-1, a human hepatic endothelioma, or Hep-3B, a liver adenocarcinoma, at any of the doses tested.

Structure of malhamensilipin A, an inhibitor of protein tyrosine kinase, from the cultured chrysophyte Poterioochromonas malhamensis.

A new chlorosulfolipid, malhamensilpin A [1] was isolated from the cultured chrysophyte Poterioochromonas malhamensis. Malhamensilipin A was demonstrated to be a modest inhibitor of pp60v-src protein tyrosine kinase. The structure was determined by detailed spectral analysis to be a novel C24 hexachloro lipid containing a vinyl sulfate ester (2,11,12,13,15,16-hexachloro-14-hydroxy-n-tetracos-1E-enol-1-sulfa te).

A mathematical model for the inhibition of the multidrug resistance-associated P-glycoprotein pump.

An extension of an earlier model of the p170 glycoprotein pump is presented. In an earlier work (Michelson and Slate, Bull. math. Biol. 54, 1023-1038, 1992), the pump was modeled using an energy-dependent model of facilitated diffusion. In this paper we add an inhibitor to the model. New equations are derived which represent either competitive or non-competitive inhibition in the pumping action of the glycoprotein. Numerical simulations were run which provide a response surface (initial loading concentration of inhibitor and its ability to compete with an ideal anti-cancer drug vs a summary measure of cytoplasmic exposure) for each scenario. The importance of the exposure profile, how it is related to ultimate tumor cell survival, and the binding requirements for developing multidrug resistance inhibitors are discussed.

Intracellular expression of P-glycoprotein in a human colon tumor cell line.

A number of human tumor cell lines originating in tissues which normally express high levels of P-glycoprotein were examined for the expression of mdr-1 RNA and P-glycoprotein and their sensitivity to doxorubicin and vincristine. There was a wide variation in expression levels and in drug sensitivity among the cell lines. In the human colon tumor cell line, LS 174T, high levels of P-glycoprotein and mdr-1 RNA were observed, but this line was very sensitive to doxorubicin and vincristine. Immunoprecipitation of P-glycoprotein from preparations of membrane and cytoplasmic proteins and immunofluorescence studies using anti-P-glycoprotein antibodies revealed that P-glycoprotein in these cells is not associated with the plasma membrane, but is, instead, found intracellularly. These results emphasize the need for functional analysis of P-glycoprotein in cells that express this mediator of multidrug resistance.

New cycloartanol sulfates from the alga Tydemania expeditionis: inhibitors of the protein tyrosine kinase pp60v-src.

Bioactivity-directed fractionation of the extracts of the green alga Tydemania expeditionis using the protein tyrosine kinase pp60v-src led to the isolation of three new cycloartanol disulfates, 1-3, which show modest inhibition of this enzyme. The structures were deduced by spectroscopic methods.

Modulation of doxorubicin efficacy in P388 leukemia following co-administration of verapamil in mini-osmotic pumps.

Co-administration of doxorubicin and verapamil in Alzet mini-osmotic pumps increased the survival of B6D2F1 mice bearing the multidrug-resistant P388/ADR leukemia. A range of doxorubicin and verapamil combinations was studied to define dose-dependent efficacy and toxicity. High doses of doxorubicin (10 mg/kg/day) and verapamil (150 mg/kg/day) could be administered alone without any effect on survival. However, combining high doses of these two agents resulted in host toxicity. Doxorubicin doses of 1 to 10 mg/kg/day in combination with verapamil at 25-100 mg/kg/day were found to improve survival compared with either agent alone. Combination therapy also improved the survival of mice bearing the drug-sensitive P388/0 leukemia when compared to anthracycline treatment alone. The efficacy of the mini-osmotic pump delivery protocol was compared with other regimens delivering the same total cumulative dose of doxorubicin via repeated i.p. injections.

Effect of verapamil on doxorubicin cardiotoxicity: altered muscle gene expression in cultured neonatal rat cardiomyocytes.

Verapamil reverses multidrug resistance acquired by cancer cells during treatment with chemotherapeutic agents such as doxorubicin by inhibiting the function of P-glycoprotein. Verapamil has also been suggested to potentiate the cardiotoxicity of doxorubicin. We have recently demonstrated that selective inhibition of cardiac muscle gene expression is among the earliest events in doxorubicin cardiotoxicity. To explore the influence of verapamil on doxorubicin cardiotoxicity, we evaluated [14C]-doxorubicin accumulation, cardiac muscle gene expression by Northern blot analysis, and ultrastructural changes in cultured cardiomyocytes in the presence and absence of verapamil. Treatment with a combination of doxorubicin and verapamil for 24 h did not augment doxorubicin accumulation in cardiomyocytes, although substantial augmentation of doxorubicin accumulation by verapamil in cardiac fibroblasts was observed. Further, treatment with verapamil for 24 h did not augment the decrease in expression of muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M isoform creatine kinase). However, we found that verapamil reduced alpha-actin gene expression in a direct, doxorubicin-independent manner. Furthermore, the effect of doxorubicin plus verapamil on alpha-actin gene expression was additive over a wide range of doxorubicin and verapamil concentrations, resulting in a selective augmentation of doxorubicin-induced inhibition of gene expression for this single muscle protein gene. This was reflected in a substantial increase in cardiac myocyte damage when treatment with verapamil and doxorubicin was compared to treatment with doxorubicin alone by thin section electron microscopy. This suggests a possible mechanism by which verapamil may potentiate doxorubicin cardiotoxicity.