RABIES CURRENT PREVENTIVE STRATEGIES. SM. Moore 2019

Rabies Current Preventive Strategies Susan M. Moore, PhD, MS, BS, HCLD(ABB), MT(ASCP)SBB

 INTRODUCTION As difficult as it is to imagine, the presence of rabid dogs in cities and the countryside was of great concern after World War II in the United States.1 The urgency of the problem led to the creation of the National Rabies Program, which began operations in 1947. The program consisted of 3 main pillars: (1) education, (2) dog control, and (3) vaccination. Relatively quickly, control of urban rabies epizootics was achieved by the early 1950s.2 This same program was endorsed by the World Health Organization (WHO)3 and resulted in successful rabies control programs in places such as Taiwan, Malaya, and Hong Kong. As early as 1983, the idea of a world rabies program was discussed by the WHO, including the cost of such a large endeavor.4 Thirty-two years later, in 2015, after years of separate efforts by veterinary health and human health organizations, a joint declaration to eliminate human deaths caused by dog-mediated rabies by the year 2030 was made by WHO, the World Organization for Animal Health (OIE), the Food and Agriculture Organization of the United Nations, and the Global Alliance for Rabies Control.5 The time to invest in ending human rabies death had arrived: rabies is a model infectious disease for the One Health approach; several pilot efforts at dog rabies control have provided proof of concept; and the United Nation’s sustainable development Goal Three targets ending epidemics of neglected tropical diseases (of which rabies is one) by 2030. Using a blueprint for each country that essentially The author has nothing to disclose. Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, KS 66502, USA E-mail address: smoore@vet.k-state.edu KEYWORDS Rabies Serology Diagnostics Disease surveillance Vaccination KEY POINTS Rabies control regulations have provided effective protection to humans and pets; however, in some areas the lack of updates in response to evolving risk situations occurs. Pet owner concerns about vaccination have led to the consideration of alterative vaccine schedules. Use of rabies serology in lieu of current recommended booster vaccination, while supported by studies, remains problematic. Vet Clin Small Anim - (2019) -–- https://doi.org/10.1016/j.cvsm.2019.02.014 vetsmall.theclinics.com 0195-5616/19/ª 2019 Elsevier Inc. All rights reserved. mimics the early 3-pillar plan of education, dog control, and vaccination, the effort is in place.6 In countries and regions that have achieved and sustained rabies control in dogs, epizootics of rabies involving other animals have become the focus. This does not mean the National Rabies Program is ended; it expands to include control of wildlife rabies, constant surveillance, and strict import regulations. Education, dog control, and vaccination is still needed to protect human from rabies exposure. The basic components of rabies control are as follows: Vaccinate pets: 70% vaccine coverage is the minimum required. Have policies and protocols for treatment of exposed pets and livestock. Have policies and procedures for animals that bite humans. Provide rabies diagnostic testing. Provide preexposure and postexposure vaccination for humans. Provide education and training for bite prevention, rabies exposure prevention, and rabies prophylaxis. Control stray dog and cat populations. Perform surveillance for rabies and maintain current epidemiology maps and information. Control rabies in wildlife.                   
LEGAL AND REGULATORY CONTROL As a global model One Health infectious disease, rabies control and prevention is guided by the WHO and the OIE.7,8 In the United States, the Compendium for Animal Rabies Control and Prevention, which contains recommendations of the National Association of State Public Health Veterinarians, is updated regularly by consideration of new/current data on rabies epidemiology, vaccines, and knowledge of the disease. For example, in 2016 the Compendium was updated to allow postexposure management of dogs, cats, and ferrets that were previously rabies-vaccinated but out of date, the same as currently vaccinated pets (Table 1).9 This change was in response to a study that demonstrated there was no significant difference in the antibody response to booster vaccination in currently vaccinated and out-of-date pets.10 Unfortunately, state and local laws and regulations, for which the Compendium recommendations act as a guide, are not always reviewed and updated in a timely Table 1 Recommendations of the National Association of State Public Health Veterinarians for rabies postexposure management of dogs and cats based on their vaccination status from the 2016 of the Compendium for Animals Rabies Control and Prevention guidelines Vaccination Status Type of Confinement Vaccinate Current Observation/Owner’s control for 45 d Booster Never vaccinated A. None: euthanize NA B. 4 mo strict quarantine: dogs and cats 6 mo strict quarantine: ferrets Vaccinate (<96 2="" 45="" 4="" a.="" a="" analyzed="" and="" are="" as="" b.="" based="" be="" been="" best="" between="" booster="" c.="" can="" cats="" certainly="" control="" current="" d="" da="" data="" date="" differences="" differing="" documented="" dogs="" epidemiology="" euthanize="" evaluation="" expected.="" exposure="" for="" from="" further="" h="" have="" however="" if="" in="" is="" it="" laws="" likely="" manner.="" mo="" moore="" na="" nbsp="" none:="" not="" observation="" obtained.="" of="" on="" other="" out="" outside="" p="" place.="" populations="" practices="" prior="" proof="" provided="" quarantine:="" quarantine="" rabies="" rates="" reasons="" regulations="" resources="" response="" results="" reviewed="" s="" serologic="" states.="" states="" strict="" surveillance="" the="" there="" this="" to="" undocumented="" updated="" updates="" vaccinate="" vaccination="" valid="" variables.="" well="" within="" wner="" years="">ASSESSING THE RISK OF RABIES Every year the rabies case data are analyzed and reported by the Rabies Section at the Centers for Disease Control and Prevention in the Journal of the American Veterinary Medical Association. 11 This information has been used to track and illustrate trends in the spread of rabies variants. Rabies variants are viral strains that circulate in reservoir species to which the virus has adapted. Vector species are mammals that are susceptible to rabies infection and are able to infect other susceptible individuals. Rabies is an RNA virus, making it susceptible to mutation and thus adaption into vector species; surveillance is meant to identify such events. An example of rabies virus crossspecies transmission occurred when positive rabies cases in skunks were reported in an area of Arizona in which skunk strain of rabies had not previously been identified.12 Viral sequencing of the positive cases determined the variant had passed from bats in the area and adapted to skunks. This demonstrates that just because an area is free of terrestrial rabies, it is not absolutely free of the risk of rabies exposure to pets and domestic animals, and hence humans. Another point to consider when assessing rabies risk in an area or region, is the “discovery” of rabies in the ferret badger population in Taiwan. Taiwan was believed to be rabies-free since 1961.13 Further study determined that rabies had been circulating in this population for years.14 Poor surveillance for rabies can create this peril by not being aware of or controlling rabies exposures from reservoir species. Both examples illustrate the key point that rabies control measures, including education and surveillance, must be sufficient and sustainable. In the Americas, all rabies variants are within the classic rabies lyssavirus species. However, companion animals travel with their owners, sometimes to areas of the world in which other lyssavirus species are present. Rabies vaccine covers many of the other lyssavirus species in phylogroup I, but those in phylogroups II and III are not.15

PREVENTING INTRODUCTION INTO RABIES-FREE AREAS Pet owners have come to view their pets as family members, so much so that pet travel is a thriving industry. When pets travel from rabies-endemic areas into rabiesfree areas, it is of primary importance that they are not incubating rabies, thereby risking the introduction of rabies into a susceptible population. The incubation period for rabies in dogs and cats is reported to be approximately 4 months.9 In the past, pets traveling or moving to rabies-free areas were subjected to long quarantines periods, established (in consideration of the incubation period) to ensure the pet was not incubating rabies. Starting in the 1990s, rabies-free areas instituted the use of rabies serology as a surrogate for protection. A defined concentration (level) of rabies virus neutralizing antibodies (RVNA), typically 0.5 IU/mL, demonstrates proof of adequate response to rabies vaccination. Once adequate vaccine response is established, a defined waiting period to account for a prevaccination infection and incubation must be achieved before the pet can enter the rabies-free area. This procedure was initiated by Hawaii in 1997 and now many rabies-free areas use similar procedures.16 Rabies 3                                                 
POSTVACCINATION SEROLOGY Studies analyzing rabies antibody level data from pets have provided information about the immune response to rabies vaccination in dogs and cats, by age, size, and number of vaccines and timing of vaccination.17–21 The major factors correlated with a robust rabies vaccine response are vaccination after maternal antibody has declined, small size, more than 1 vaccination, and time interval between vaccination and blood draw of 15 to 30 days. Evidence from previous studies has indicated that type of vaccine (multivalent vs monovalent, and manufacturer) can also play a role.17,18Rabies serology results from The Kansas State University (KSU) Rabies Laboratory from June 2015 to July 2017 were used to evaluate factors affecting RVNA levels in dogs. Evaluation of RVNA levels from 2 groups of pet dogs, that is, dogs being prepared to travel to rabies-free areas (export group) and dogs whose owners prefer to check rabies titer rather than revaccinate (core vaccine group), demonstrated that timing of blood sampling influences the probability that the pet was adequately vaccinated (Fig. 1). In the export group, pet owners and their veterinarians were highly motivated to test at the expected time interval to coincide with the peak response (15–30 days after vaccination). In this group, only 4.5% of the dogs had a result less than 0.5 IU/mL, compared with 16.1% of in the core vaccine group. The proportion of dogs with inadequate rabies antibody levels in the core vaccine group was similar to the proportion of dogs with nonprotective antibody levels to other core vaccines, at 18%, 13%, and 14% for canine distemper, canine adenovirus, and canine parvovirus, respectively. A repeated observation in these studies is the higher probability of failure to mount or sustain a robust RVNA response in young animals. The presence of maternal antibodies has been identified as one reason for this finding, due to interference by maternally derived rabies with vaccine antigen presentation to the immune system.22,23 However, the decline of the primary humoral immune response to below detectable levels before 6 months of age is also a factor. Both duration and magnitude of response is affected by the number of vaccinations administered.20 The influence of age on the probability of inadequate response to rabies vaccination is consistent, even when data are stratified by dog size. Results of rabies serology testing of 20,447 dogs being prepared for travel to rabies-free areas, performed at KSU Rabies Laboratory from July 2016 to April 2017, demonstrated the influence of age by breed size (Fig. 2). Breed size was defined by American Kennel Club standards. The largest group that failed to respond to vaccination was younger than 2 years (48.5% <2 1.="" 19.5="" 1="" 2015="" 2017.="" 29.0="" 4="" 6="" a="" age="" an="" and="" antibody="" are="" areas.="" at="" be="" because="" being="" between="" booster="" brought="" challenge="" check="" combined="" compared="" considered="" core="" correlation="" could="" dogs="" export="" exposure.="" fig.="" findings="" first="" for="" from="" give="" has="" having="" in="" inadequate="" increase="" increased="" into="" july="" june="" know="" ksu="" level="" months="" moore="" nbsp="" of="" p="" performed="" pets="" positive="" practice="" prepared="" probability="" proportion="" providing="" question="" rabies-free="" rabies="" response="" risk="" rvna="" serology="" studies="" survival="" than="" that="" the="" these="" titer="" to="" until="" vaccination.="" vaccination="" vaccine="" waiting="" we="" with="" year="" years="" younger="">VACCINATION CONCERNS BY PET OWNERS AND VETERINARIANS Rabies control in the United States has been so effective that the canine variant was eliminated from the United States in 2007.25 This achievement is also a challenge for maintaining a sufficient level of awareness of the continued risk of rabies exposure, particularly lowering awareness of the importance of rabies vaccination by pet owners. In addition, an increasing focus on the human-animal bond has changed how pet vaccination is evaluated and consequently, is either valued or questioned. Concerns regarding adverse reactions and immune modulation caused by vaccination have increased among pet owners and some veterinarians, who have suggested the use of smaller doses of rabies vaccine to potentially reduce reaction rates, and using rabies serology to provide proof of immunity and thus waive routine booster vaccinations. The frequency of reactions to rabies vaccines has been evaluated by industry groups and organizations. The following are potential vaccine adverse reactions in dogs and cats according to the American Animal Hospital Association Canine Vaccine Guidelines, 201726 and the American Association of Feline Practitioners, Feline Vaccination Advisory Panel Report, 2013,27 respectively: Injection-site reactions Allergic or immune-mediated reactions Tumorigenesis Vaccine-induced immunosuppression Anaphylaxis Injection-site sarcomas A 2007 study of approximately 1.2 million dogs whose medical records were in a large veterinary database reported that the rate of vaccine-associated adverse events Fig. 2. Percentage of dogs with inadequate rabies vaccine response by age (in years) and breed size group, from a data set of 20,447 dogs tested for rabies antibody at KSU from July 2016 to April 2017. Rabies 5 (VAAE) within 3 days of vaccination in d   ogs was 38.2 of 10,000 dogs vaccinated.28 A study of VAAE within 30 days of vaccination in almost 500,000 cats in the same database and performed by the same group reported a VAAE rate of 51.6 of 10,000 cats vaccinated; 92.0% were diagnosed within 3 days after vaccination. Anaphylaxis comprised 17 of 2560 total cases of feline VAAE (0.7%).29 These organizations recognize that adverse reactions are likely to be underreported by both veterinarians and pet owners27 and that in veterinary medicine there is no requirement to report VAAE, either known or suspected.26                                                                                                                                 
RABIES VACCINE REGIMENS Fear of vaccine adverse reaction/immune modulation or questioning the need for vaccination among pet owners parallels the similar concerns for human vaccination that has existed for many decades. It may be useful to compare rabies vaccines and vaccine regimens for humans and pets for perspective. For humans who are at increased risk of rabies exposure and thus rabies preexposure vaccinated with a series of 3 vaccinations,30 2 booster vaccines are given at day 0 and day 3 on exposure, regardless of titer. Titer does not affect the recommendations for people because it is absolutely necessary to stimulate an anamnestic response. This is to ensure (1) the highest level of RVNA is present to neutralize the virus; and (2) that other immune system components are stimulated (T memory cells), to help with B-cell antibody production and cytokine responses, which alert immune effectors to the threat. For animals that are preexposure vaccinated, after a known or suspected rabies exposure, regardless of titer, a single booster vaccine is given on day 0 (or as soon as seen by a veterinarian), for the same reasons as stated previously. One vaccine booster is administered rather than 2, because to ensure effective prevention in humans for a fatal disease such as rabies, no risk of understimulating the anamnestic response is tolerated. However, for animals, protection from rabies is intended to prevent rabies exposure to humans; thus, different standards, different regimens/policies are applied. Humans who have had preexposure vaccination and continue to be at risk of rabies exposure due to occupation or travel are recommended by the Advisory Committee on Immunization Practices (ACIP) and WHO to have routine titer checks to ensure an adequate level exists to ensure a fast, robust rise in immune defenses on postexposure vaccinations, and to protect from unrecognized exposures.7,31 In addition, humans who have been preexposure vaccinated are informed to recognize rabies exposure and to seek postexposure treatment, including wound care, which alone can reduce the chance of infection by up to 60%,32,33 whereas pets cannot. Although rare, vaccinated pets have succumbed to rabies after exposure to rabid animals.34,35 The dose of human rabies vaccine used is related to the antigenic content and the route. Human rabies vaccines must provide 2.5 IU antigen by intramuscular (IM) injection, whether in 1 mL or in 0.5 mL of diluent. An intradermal (ID) dose uses a tenth of the antigen in an IM dose and can be given for preexposure and postexposure rabies vaccination. Although ID rabies vaccination is not approved in the United States, it is recognized by the WHO. Some rabies ID regimens require multiple ID injections per day at different sites on the body.7 Horses, cows, and pigs typically receive a 2-mL dose, and dogs and cats, 1 mL; the entire list of US-approved veterinary vaccines are listed in Compendium for Animals Rabies Control and Prevention.9 As with human vaccines, all rabies vaccines must meet minimum standards for safety, efficacy, and immunogenicity. The Code of Federal Regulations (CFR) defines standard requirements for both human and animal vaccines.36 6 Moore                                                                                                                                                                                                                                                                   THE IMMUNE RESPONSE TO RABIES VACCINATION Vaccines must contain sufficient antigen to induce an adequate immune response in the target species that affords protection from infection, disease, or in some cases, reduction in severity of disease. Vaccines induce immune responses based on amount of antigen and the corresponding immune cell receptor availability and degree of specificity (including avidity and affinity). Receptor specificity is controlled by the major histocompatibility complex (MHC), which is polygenic, meaning there is a great diversity of MHC genes and hence molecules within a species. MHC molecules on the surface of immune cells take up, process, and present antigen to immune cells for the induction immune response. Alongside the diversity of immunity induced by MHC molecules, there is variation between animals in a population in terms of protective response. A vaccine must show the ability to produce a robust and protective immune response among representative animals of the intended species. The notion that size of animal is the major factor in vaccine response, with larger animals mounting a lower response than smaller animals for a defined antigenic dose, or that smaller animals require a smaller dose is prevalent among those who fear the effects of vaccination. The role of immune genetics and mechanisms of the immune response, as described previously, argues against this. Moreover, further analysis of the KSU study of the canine RVNA response (see Fig. 2), by breed and by size, indicated that breed also influences the probability of mounting an adequate RVNA response to vaccination (Fig. 3). In both the 10 breeds with the highest percentage of dogs with inadequate RVNA (<0 .5="" 10="" 1="" 2016="" 2017.="" 20="" 3.="" 3="" 7="" a="" adults="" all="" alone.="" also="" although="" and="" antibody="" april="" are="" as="" associated="" associations="" at="" average="" bar="" based="" be="" been="" breed="" breeds="" by="" case.="" cats="" chart="" children="" colleagues="" data="" demonstrated="" difficult="" diluted="" displaying="" distribution="" dog="" dogs.="" dogs="" dose="" doses="" dosing="" each="" entire="" factor.="" fig.="" findings="" for="" from="" gene="" giant="" group="" groups="" has="" highest="" however="" human="" in="" inadequate="" indicating="" infants="" into="" is="" it="" iu="" july="" kennedy="" ksu="" large="" level="" levels.37="" lowest="" medium="" mhc="" ml="" moreover="" nbsp="" no="" not="" of="" on="" other="" p="" percentage.="" percentage="" pie="" rabies="" ranking="" receive="" recommend="" reduced="" represented.="" represented="" response.="" response="" result="" rvna="" same="" set="" shown="" size="" small="" some="" study.="" study="" supported="" tested="" that="" the="" there="" these="" this="" to="" toy="" vaccine.="" vaccine="" vaccines="" was="" were="" who="" with="" within="" would="">
TITER TESTING TO INFORM VACCINATION DECISIONS One way to address concerns over vaccine adverse reactions/immune modulation is allowance of RVNA titer checks in lieu of routine vaccination. Indeed, in general, revaccination of an animal already protected does not result in enhanced disease resistance. However, the main challenge for a particular disease or infection for which vaccination is available is to provide proof of “already protected.” Peer-reviewed data regarding proof of rabies protection afforded by vaccination, as well as the predictive ability of rabies serology results, is primarily obtained from published rabies challenge studies. Per the CFR, studies for vaccine approval must demonstrate protection by survival of the challenged animals; included in the requirement is proof of immunogenicity by the measurement of rabies antibody levels.36 The following list includes information from challenge studies publications,24,38,39 the 9 CFR 113.209,36 and the Compendium,9 regarding rabies vaccines: Vaccines for dogs and cats: At least 86% to 87% of animals must survive challenge and at least 80% of unvaccinated controls must succumb to challenge. Current rabies vaccines are licensed for 1 to 3 years. Dogs and cats with rabies serology results greater than 0.5 IU/mL survive more frequently than dogs and cats with results less than 0.5 IU/mL; there are almost 100% survival rates in animals with RVNA greater than 0.5 IU/mL. There are a few reported animals with an RVNA result greater than 0.5 IU/mL that succumbed to challenge. Challenge studies require experimental challenge through the intracerebral route, which is considered an extreme challenge route of exposure for pets. Rabies serology methods have a precision (CV%) of 50% (or less), meaning an RVNA level of 0.5 IU/mL could actually be 0.25 to 1.0 IU/mL in any given assay run. Greater or lesser variability may occur between laboratories and assay types. The 0.5 IU/mL “adequate level” was identified as robust based on this knowledge. In the conclusions made by Aubert38 in a 1992 publication presenting a comprehensive review of challenge studies in dogs and cats, it was stated “The security of the protection constituted by this threshold [RVNA level] would be increased by the extent to which it exceeds the level recognized as effective against experimental challenge in cats and dogs 0.1 IU/mL and 0.2 IU/mL, respectively, measured by the RFFIT” (rapid fluorescent focus inhibition test). Even before this study was published, a study by Bunn and Ridpath24 (1984) analyzed the probability of survival by RFFIT level using challenge study data and concluded that survival rate increased as RVNA level increased up to approximately 1.0 IU/mL, with a survival probability of approximately 99% at level of 0.5 IU/mL. Whether these findings can be extrapolated to pets without current vaccination status is the question at hand. 8 Moore                                                                                 
 IMMUNOLOGIC PROTECTION IN THE FACE OF EXPOSURE Currently, limited published studies provide rabies serology to correlate with survival data in dogs and cats. A study by Lawson and Crawley40 provides some information. The investigators challenged vaccinated dogs and cats at 5 and 4 years, respectively, after vaccination and reported that 92% of dogs and 100% of cats survived; 54% of dogs and 87% of cats had detectable RVNA before challenge. Assuming that immune protection extends for an indeterminate period beyond the due date for a booster is reasonable, based on immunologic principles. An argument supporting this idea is that for human postexposure prophylaxis (PEP), a single dose (40 IU/kg bodyweight) of heterologous antirabies serum was protective in combination with vaccination.41 In the case of an unrecognized rabies exposure in a pet, it is assumed that stimulation of memory immune cells to become effector cells would provide protection, rather than vaccination. There is evidence of this kind of response in a report of a human organ recipient of a rabies-infected liver, who survived even though their last rabies vaccination was many years previously,42 due to a robust protective anamnestic response. However, this was a single case, and the outcome cannot be reliably generalized to larger groups, as exposures and the probability of infection can vary considerably.32 PEPs and other protocols are based on the level of risk regulatory authorities and policy makers consider acceptable. 
SEROLOGIC DATA TO DEMONSTRATE PROTECTION AGAINST RABIES Based on the data from challenge studies, there is a valid argument for use of rabies serology to estimate rabies protection. However, several critical parameters need to be defined before confidently using rabies serology as a correlate of protection: Definition of an adequate antibody response (correlation of protection level) Significance of a rise or fall in antibody level Method (assay used) of measurement (correlate with protection, with ample data) Reported value: IU/mL or titer Timing of blood sampling and how often is the level assessed 4 weeks after vaccination? Yearly? After exposure to rabies? Will there be different acceptance levels based on timing of blood draws? Approval for laboratories providing testing; routine proficiency testing required for approval (as for rabies serology for pet travel and equine infectious anemia/ Coggins for horses). In an article that attempted to define rabies antibody level as proof of protection in different wildlife species using rabies serology results, few conclusions could be drawn from a systematic review of published studies, due to the highly variable study designs and results.39 Also, in this article, sets of serum samples from challenge studies in dogs, foxes, skunks, raccoons, raccoon dogs, and mongooses were tested using 2 types of assay: serum neutralization (RFFIT) and a blocking enzyme-linked immunosorbent assay (ELISA). it was concluded that a level of 0.25 IU/mL and 40% inhibition by RFFIT and ELISA, respectively, were reasonably associated with survival; however, species differences were noted. Similarly, a study comparing RFFIT results with indirect ELISA results for a human rabies vaccine trial demonstrated that results from the assays were variously correlated by time since vaccination; results at day 14 were very poorly correlated, whereas results at day 90 had good correlation.43 Although there are studies that have demonstrated good correlation between serum neutralization and ELISA techniques for rabies antibody measurement,44–46 it is necessary to establish requirements by method validation, including diagnostic Rabies 9 specificity and sensitivity, before approval of an assay for a specific use, such a proof of adequate vaccine response in lieu of booster vaccination.47                                                                                                                                                                                                                                                                                       PRACTICAL USE OF TITER TESTING The use of serology for verification of rabies immunity should be reserved for wellvaccinated pets (dogs and cats that have received a primary vaccination, followed by 1 or 2 boosters). Rabies titer checks in animals could also be used for prospective serologic monitoring to help determine whether an animal has been previously vaccinated.9 In addition, they can be used if public health officials need to determine the risk of an exposed pet becoming infected after a nonstandard postexposure treatment or other unusual circumstances (eg, vaccinated animal for which no licensed vaccine is approved, such as an alpaca or monkey), or in pets for which routine vaccinations are contraindicated because of health concerns.48 In a perfect world, defining pet risk would also be part of the decision to perform titer testing.         
ADVANTAGES AND DISADVANTAGES OF USING SEROLOGY IN LIEU OF REVACCINATION Advantages Reduces the number of vaccinations, hence reduces the risk of VAAEs. If required yearly for pet licensing, would identify pets that fail to respond to vaccination or have low titers, making them at higher risk of vaccine failure on rabies exposure. Disadvantages The accuracy of the RVNA level to predict protection in pets for which vaccination is out of date is unknown because there have not been any studies to determine this. Routine vaccination stimulates/activates both humoral and cellular immune effectors. Enhanced assurance of human protection to humans is achieved by routine vaccination of pets, based on current knowledge.

SUMMARY Current measures to prevent and control rabies in animals work very well, but there are challenges, such as decreased awareness of rabies risks, given the shift of rabies cases from primarily dogs before the establishment of the National Rabies Program in the 1940s, to wildlife. The threat of rabies virus variants adapting to new wildlife species is real, requiring continuous surveillance and prevention procedures. Viewpoints and opinions toward vaccination of pets and the compilation of robust data correlating rabies serology with adequate protection will drive changes in rabies control policies. An RVNA level of 0.5 IU/mL in a dog or cat demonstrates a continuing robust response to rabies vaccination and there is an expectation of sufficient immunologic memory to produce a protective response following exposure. Ideally, the animal would also receive postexposure treatment (wound care/cleaning and booster vaccination). Without postexposure treatment, complete protection is somewhere between expected and probable, but is unknown at this time, given lack of published data and variability in immune status at the time of exposure and exposure type. No vaccine can be 100% effective in every animal because of these variables. Given the importance of rabies protection in pets to human protection, an abundance of caution regarding defining the protective level and how it is measured in pets is warranted. 10 Moore Rabies serology testing is not regulated, except for the recommendation of the ACIP to measure RVNA by the RFFIT. Experts in public health administration are responsible for weighing the risks of recognizing RVNA levels in animals solely as proof of rabies protection. Science is just one part of the equation; public health decisions in light of scientific data is another, and a multitude of factors, including the enforcement of laws both technically and politically, must be considered.                                                   
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VIRUS Y CULTIVOS CELULARES. Patricio Berríos Etchegaray. 2019

VIRUS Y CULTIVOS CELULARES

Patricio Berríos Etchegaray

2019

El cultivo celular es el proceso mediante el que células, ya sean células procariotas o eucariotas, pueden cultivarse en condiciones controladas. En la práctica el término "cultivo celular" se usa normalmente en referencia al cultivo de células aisladas de eucariotas pluricelulares, especialmente células animales.

La implementación de los cultivos celulares hizo posible el desarrollo de la virología. El descubrimiento de los antibióticos permitió utilizar el cultivo celular “in vitro” como una técnica de laboratorio de rutina. Desde 1949 en que Enders descubrió que el virus de la poliomielitis podía multiplicarse en cultivos celulares casi todos los virus animales han sido propagados en cultivos celulares. Por otra parte, se acepta que cualquier tipo de células de mamíferos, capaces de dividirse “in vivo”, lo pueden hacer “in vitro”. 

En los inicios de la virología los virus se multiplicaban en animales susceptibles generalmente animales de laboratorio, y en huevos embrionados de gallina.  El amplio uso de los cultivos celulares en la propagación de virus animales ha permitido no depender de la utilización de animales de laboratorio para este fin.

Se conocen tres modalidades de cultivos celulares: cultivo de órganos, de tejidos y de células. Cultivo de órganos: Trozos pequeños menores de 1 cm de diámetro pueden mantenerse “in vitro” durante 7 a 14 días sin perder su estructura y función. Su aplicación más importante ha sido el cultivo de trozos de mucosa respiratoria para realizar estudios de histopatogénesis causados por virus respiratorios. En cultivos de tejidos se utilizan fragmentos de tejidos finamente picados y embebidos en plasma lo que les permite adherirse al vidrio de las botellas de cultivo.  En los cultivos celulares el tejido se disocia mecánicamente o mediante enzimas proteolíticas como la tripsina al 0,25%. Una vez contadas las células y suspendidas en un medio nutritivo adecuado se siembran en una concentración adecuada (100.000 células por 1 ml) en botellas, tubos o microplacas. Para multiplicarse “in vitro” las células necesitan un medio de cultivo adecuado que le proporcione los nutrientes necesarios para multiplicarse. Uno de los medios más utilizados es el medio Eagle esencial (MEM) que es básicamente una solución isotónica de sales, tamponado a un pH de 7,4, que contiene glucosa, vitaminas, coenzimas y aminoácidos. A los medios de cultivos de células es necesario agregarles antibióticos para impedir la infección bacteriana, y suero fetal bovino que contiene un factor de crecimiento.  En este medio y a una temperatura de 37° C las células se multiplican sobre la superficie del vidrio del continente formando monoestratos o monocapas de células visibles con un microscopio invertido.  Ejemplo: el virus herpes bovino 1 se mutiplica en células de riñón fetal bovino produciendo un típico efecto con destrucción de las células y la liberación de virus al medio de cultivo.

Considerando el número de divisiones que las células son capaces de realizar “in vitro” se describen tres tipos de cultivo: Cultivo celular primario en que las células solo se dividen en un número bajo, unas 20 a 30 veces, conocidos como pasajes o subcultivos. En los cultivos celulares secundarios las células pueden dividirse unas 100 veces. Las líneas celulares pueden dividirse potencialmente en forma indefinida debido a que han perdido la inhibición por contacto. Estas líneas se originan desde tumores o por mutaciones.  Ejemplos de líneas celulares son las células HeLa provenientes de un cáncer uterino, y las células MDBK (Madin-Darby bovine kidney).

Las células en un cultivo celular pueden presentar dos tipos de morfologías: Tipo fibroblasto que se originan desde tejidos conectivos y tienen forma de huso. Tipo epiteliales que se origina desde órganos glandulares y tienen forma poligonal.

El efecto que pueden causar los virus inoculados en cultivos celulares depende del tipo de virus y de la susceptibilidad de las células. Este efecto puede ser: efecto citopático (ECP) lítico y muerte celular debido a la detención de la síntesis de macromoléculas tales como ácidos nucleicos y proteínas celulares, a alteraciones de la permeabilidad de los lisosomas, a alteraciones de la membrana celular, inducción de aberraciones cromosómicas y desarrollo de cuerpos de inclusión que alteran la estructura y función de las células infectadas. En las infecciones no citolíticas no hay alteraciones morfológicas ni en la división celular. En la transformación celular ciertos virus oncogénicos, que no destruyen a las células infectadas, las transforman al integrar su genoma en el genoma celular, causando diversos efectos como la pérdida de la inhibición por contacto lo que las hace dividirse indefinidamente.

Mis experiencias con cultivos celulares

En 1973, en Davis University of California USA, me inicié en los cultivos celulares al estilo americano. La tecnóloga del Dr Delbert McKercher, Midori Ethel Wada que era Master of Science al pedirle ayuda para hacer cultivos celulares me dijo aquí los candidatos a doctores se hacen sus cositas solitos y me pasó un pequeño manual del laboratorio que contenía todos los pasos para realizar un cultivo celular. Empecé haciendo cultivos de riñón fetal caprino necesarios para propagar el nuevo virus herpes caprino motivo de mi tesis doctoral. Me costó, hasta que aprendí las mañas para hacer un buen cultivo celular. El virus herpes producía un nítido efecto citopático lo que facilitaba su observación. Solo tuve un problema cuando se me contaminaron los cultivos y no podía encontrar el origen de la contaminación. Le busqué por todos lados y los hongos contaminantes se mantenían, y yo estaba contra el tiempo… desesperado porque se me iba la tesis y el doctorado,  un día estaba absorto mirando la cámara de cultivos celulares y me fijé en un frasquito que estaba al fondo de la cámara, era un frasco de vidrio color café que contenía NaOH para alcalinizar los medios ácidos, y empecé a hablar conmigo mismo estarán lo hongos allí y mi mentalidad científica me decía que no, porque los hongos no crecen en ese pH tan alcalino, mi otro yo el discutidor cuestionaba la situación argumentando que los hongos crecen en cualquier parte. Lo sopesé y eliminé el hidróxido, preparé uno nuevo y lo esterilicé. ¡Y se fueron los hongos! Seguí con mi trabajo entregando el virus herpes clonado tres veces a su debido tiempo para ser inoculado en cabras gestantes lo que fue un éxito al producir aborto. La tesis doctoral fue aprobada en 1974 sin mayores problemas.

En 1976 empecé a hacer cultivos celulares en el laboratorio de virología de la Escuela de Medicina Veterinaria de la U de Chile. Necesitaba células de origen equino para estudiar virus respiratorios equinos especialmente el virus de la rinoneumonitis equina. Mi alumno de tesis era Víctor Riveros V. hombre versátil, buen clínico y bueno para el laboratorio. Pasó un mes sin conseguir que creciera una sola célula de riñón fetal equino. Probamos el medio de cultivo, el suero fetal, el agua  destilada y no se replicaban ante la paciencia de mi alumno de tesis. Tuve una inspiración y eliminé todo, absolutamente todo, y preparamos todos los medios de nuevo. Y las células crecieron. Era el agua destilada la mala.  De ahí en adelante todo bien dentro de la normalidad de los cultivos celulares. Fueron muchos mis alumnos tesistas que trabajaron las células con éxito. Incluso Francisco Cortes C.  en 1982 llegó más allá y por iniciativa propia hizo pasajes del riñón fetal equino hasta alcanzar un número mínimo que permitía considerarlas como una línea celular establecida la que fue estudiada en la tesis de Aldo Gaggero B. “Caracterización de una línea celular de crecimiento “in vitro” derivada de riñó fetal equino” 1984. La línea celular yo la denominé RFE-13.

Tuve éxito en hacer cultivos primarios de riñón fetal bovino, equino y caprino, los de porcino nunca me resultaron. Cultivos de células testiculares de caprino las utilicé en mi tesis doctoral.

Siempre amé a mis células, les hablaba y las trataba con una gran consideración como si fueran mujeres. No me fallaban. Estos cultivos eran exigentes, no sabían de feriados. El suero fetal había que ir a buscarlo al matadero Lo Valledor bien temprano en la mañana. Su filtración era demorosa, pero así se ahorraba porque el suero fetal de afuera era carísimo. Por cierto los riñones fetales había que ir a buscarlos al matadero. Un día me descresté en una escalera resbalosa y perdí unos buenos litros de suero fetal bovino. Otra vez casi choque en mi auto que resbaló en el petróleo que se había derramado en la carretera. Siempre pasaban cosas, recuerdo cuando un viejito me lavó con agua no limpia mis riñones, yo se lo agradecí por su buena intención y después los tuve que eliminar porque estaban contaminados. Hasta la política influyó en la mantención de las células, cuando había protestas contra Pinochet se cotaba la luz con los cadenazos contra los cables de la corriente, y se apagaban los congeladores causando la muerte de las células.

En realidad fue una verdadera odisea iniciar el cultivo celular en mi laboratorio de Virología, sin medios económicos, sin personal adecuado, y sin la comprensión de algunas autoridades universitarias que no entendían las dificultades que presentaba esta técnica. Recuerdo a un colega austríaco que nos visitó en 1976 y al ver mi laboratorio me dijo que así se trabajaba en los inicios de la virología, sin tener congeladores de -70° C necesarios para mantener a las células sin tener que hacer pasajes todas las semanas como estábamos obligados a hacerlo.

Actualmente la cosa es diferente, hay congeladores de -70° C., y Nitrógeno líquido para mantener las células, el suero fetal se importa al igual que los medios de cultivo. Hay cámaras de cultivo de alta complejidad.  Microplacas y pistolas ACCU-JETT llamadas auxiliares de pipetas, todo disponible en el mercado. Obviamente que para comprar estos artefactos hay que tener financiamiento con algún proyecto de investigación que lo considere.

Echo de menos a los cultivos celulares porque eran cosas vivas que requerían un cuidado extremado y mucho cariño. Actualmente aconsejo a mi señora, Damaris Vega P.  que trabaja con cultivos celulares de peces en la escuela de Medicina Veterinaria de la U. de Chile.