Vaccination of Tunisian dogs with the lyophilised SAG2 oral rabies vaccine incorporated into the DBL2 dog bait

The protective effect of the lyophilised SAG2 oral vaccine bait DBL2, already demonstrated on laboratory dogs, needed to be verified on common Tunisian dogs. Seven Tunisian dogs consumed totally or partially one DBL2 bait containing 10(8.3) TCID50 of the highly attenuated rabies vaccine strain, SAG2. Five of the seven vaccinated animals survived a challenge administered 33 days later with a Tunisian canine street rabies virus to which five of the six controls that were not vaccinated and had no specific antibodies succumbed. The partial or total consumption of a single DBL2 bait thus conferred a protective immune response similar to that observed in laboratory dogs to dogs of poor health status. The sero-antibody response was, however, weak: only two vaccinated dogs exhibited a significant neutralising antibody response after vaccination and before the challenge, and four after the challenge.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.     

Oral immunization of foxes with avirulent rabies virus mutants

SAG1, a rabies virus strain bearing one mutation which abolishes virulence for adult animals, was constructed from the SADBern strain of rabies virus which has previously been used as live vaccine for oral immunization of foxes. SAG1 also bears an antigenic mutation which serves as an additional marker of the strain. Studies on mice and four species of wild rodents showed that SAG1 is totally avirulent whereas SADBern is still pathogenic after intracerebral, intramuscular or oral inoculation and thus could cause cases of rabies. Trials of oral vaccination performed on foxes with SAG1 indicate that it is as effective as SADBern. The SAG1 strain represents a significant progress in the search for an efficient and safe live rabies for the oral immunization of wild animals.     

Experimentally induced rabies in four cats inoculated with a rabies virus isolated from a bat

Four cats were inoculated IM with rabies virus isolated from the salivary gland of a naturally infected big brown bat (Eptesicus fuscus). The 4 cats developed clinical signs of rabies after a median incubation period of 42 days. The median duration of clinical illness was 5 days. Results of fluorescent antibody evaluation, mouse inoculation, and tissue culture isolation indicated large differences in virus concentrations in various areas of the CNS of individual cats. These differences also were observed between cats. Rabies virus was isolated from the salivary glands and saliva of 2 cats; urinary bladder was the only other nonneural tissue found infected. Our observations indicated that cat rabies can be caused by bat rabies virus; that cats thus infected have infectious saliva during aggressive behavior and can therefore transmit the disease; and that adequate specimens of hippocampus, cerebellum, and brain stem are essential for reliable determination of rabies infection. The findings support recommendations for regular rabies vaccination of cats, even in areas of rabies-free terrestrial mammals.     

Safety and immunogenicity of a vaccine bait containing ERA strain of attenuated rabies virus.

Ninety percent of foxes fed commercial ERA vaccine in a specially designed bait developed rabies serum neutralizing antibodies. The vaccine bait did not cause clinical signs of rabies when consumed by foxes, raccoons, skunks, dogs, cats, cattle and monkeys. When presented, in the laboratory, to wild rodents of the species Microtus, Mus musculus and Peromyscus, the vaccine baits caused vaccine-induced rabies only in Mus musculus. Laboratory mice of the CD-1 and CLL strain were susceptible to vaccine-induced rabies; however, studies showed that transmission of virus to other animals did not occur. These studies suggest that the vaccine bait described could be useful in a rabies control program in areas where foxes and wild dogs are the principal vectors.     

A pathological study of the salivary glands of rabid dogs in the Philippines

Rabies is a zoonotic disease caused by the rabies virus. While the salivary glands are important as exit and propagation sites for the rabies virus, the mechanisms of rabies excretion remain unclear. Here, we investigated the histopathology of the salivary glands of rabid dogs and analyzed the mechanism of excretion into the oral cavity. Mandibular and parotid glands of 22 rabid dogs and three control dogs were used. Mild to moderate non-suppurative sialadenitis was observed in the mandibular glands of 19 of the 22 dogs, characterized by loss of acinar epithelium and infiltration by lymphoplasmacytic cells. Viral antigens were detected in the mucous acinar epithelium, ganglion neurons and myoepithelium. Acinar epithelium and lymphocytes were positive for anti-caspase-3 antibodies and TUNEL staining. In contrast, no notable findings were observed in the ductal epithelial cells and serous demilune. In the parotid gland, the acinar cells, myoepithelium and ductal epithelium all tested negative. These findings confirmed the path through which the rabies virus descends along the facial nerve after proliferation in the brain to reach the ganglion neurons of the mandibular gland, subsequently traveling to the acinar epithelium via the salivary gland myoepithelium. Furthermore, the observation that nerve endings passing through the myoepithelium were absent from the ductal system suggested that viral proliferation and cytotoxicity could not occur there, ensuring that secretions containing the virus are efficiently excreted into the oral cavity.     

The ERA Strain of Rabies Vaccine

An antigenic extinction trial in cats showed that the ERA rabies vaccine had superior antigenic properties over Flury H.E.P. C.E.O. and killed tissue culture rabies vaccine.

Dogs and cats on a duration of immunity study of ERA rabies vaccine were challenged with fox salivary gland “street” rabies virus. The results of this challenge show a duration of immunity of five years in dogs and four years in cats.

Vaccination of dams in late pregnancy with ERA rabies vaccine resulted in transference of maternal antibody to the newborn, in both cattle and dogs. This maternally derived antibody interfered with the successful active immunization of the young calf. Calves free of antibodies for rabies could be successfully vaccinated as early as 17 days of age and were able to withstand a challenge with virulent “street” rabies virus two years later.

     

Twenty year experience of the oral rabies vaccine SAG2 in wildlife: a global review

The SAG2 vaccine (RABIGEN® SAG2) is a modified live attenuated rabies virus vaccine, selected from the SAD Bern strain in a two-step process of amino acid mutation using neutralizing monoclonal antibodies. The strain is genetically stable and does not spread in vivo or induce a persistent infection. Its absence of residual pathogenicity was extensively demonstrated in multiple target and non target species (such as wild carnivores and rodent species), including non-human primates. The efficacy of SAG2 baits was demonstrated according to the EU requirements for the red fox and raccoon dog. The use of safe and potent rabies vaccines such as SAG2 largely contributed to the elimination of rabies in Estonia, France, Italy and Switzerland. Importantly, these countries were declared free of rabies after few years of oral vaccination campaigns with SAG2 baits distributed with an appropriate strategy. The excellent tolerance of the SAG2 vaccine has been confirmed in the field since its first use in 1993. No safety issues have been reported, and in particular no vaccine-induced rabies cases were diagnosed, after the distribution of more than 20 million SAG2 baits in Europe.     

Susceptibility of domestic dogs and cats to Australian bat lyssavirus (ABLV)

The susceptibility of cats and dogs to Australian bat lyssavirus (ABLV; genotype VII) was investigated by intramuscular (IM) inoculation of 10(3.7)-10(5) 50% tissue culture infective doses (TCID(50)) of virus followed by observation of experimental animals for up to 3 months post-inoculation (pi). Each experiment also included positive and negative controls, animals inoculated with a bat variant of rabies virus (Eptesicus I, genotype I), or a 10% suspension of uninfected mouse brain, respectively. Each of the ABLV-inoculated cats showed occasional abnormal clinical signs, but none died. Necropsies performed at 3 months pi revealed no lesions, and no viral antigen, in the central nervous system of any cat. ABLV could not be recovered from any cats. However, rabies virus-neutralizing antibodies were detected between 4 and 14 weeks pi in the sera of all three ABLV-inoculated cats. At 2-3 weeks pi, three of the five ABLV-inoculated dogs showed very mild abnormal clinical signs that persisted for 1-2 days, after which the dogs recovered. At 3 months pi, when all dogs were necropsied, neither lesions nor ABLV antigen were detected in, and virus was not isolated from, any dog. No ABLV RNA was detected by polymerase chain reaction (PCR) in clinical or necropsy samples from the three ABLV-affected dogs. However, all ABLV-inoculated dogs seroconverted by 2 weeks pi, and serum antibody titres were higher than those observed in cats. CSF, collected at 3 months pi, was positive for rabies virus-neutralizing antibody in two ABLV-inoculated dogs.     

Efficient distribution of oral vaccines examined by infrared triggered camera for advancing the control of raccoon dog rabies in South Korea

The field distribution of the oral rabies vaccine is effective in controlling the spread of rabies. The present study aimed to investigate efficient distribution locations based on the environment, contact rate, and consumption by target wildlife species in South Korea. The target species (Korean raccoon dogs, domestic dogs, and feral cats) accounted for 945 contacts (52.2%), in total 1,808 contacts. There were 863 (47.8%) contacts by non-target species. Raccoon dogs, a main reservoir of rabies in South Korea, had the highest contact rate (34.1%) among all species. The contact rate by target species was highest at riparian sites and bushy mountainous vegetation, where raccoon dogs are abundant. There was remarkable contact by raccoon dogs in mountainous areas below 150 m with bushy vegetation. Our results indicate that these locations are efficient areas for vaccine distribution, especially targeting the raccoon dog. Vaccines were continuously contacted with intervals ranging from one hour to one day. Vaccines at 94.4% of the distribution points were completely consumed within two weeks. The mean consumption rate was 95.2 ± 1.93% during the overall study period. These findings suggest that the oral rabies vaccine attracts wildlife including domestic dogs and feral cats. Our results suggest that low sections of mountainous areas with bushy vegetation and/or neighboring riparian areas are rich in target wildlife species (especially raccoon dogs) and are efficient locations for vaccine distribution to control rabies in South Korea.     

Use of monoclonal antibodies to confirm vaccine-induced rabies in ten dogs, two cats, and one fox

A panel of 8 monoclonal antibodies to rabies glycoprotein antigen was used to characterize the modified-live virus vaccines marketed in the United States during the last 10 years. Thirteen of 14 rabies virus isolates from 11 dogs, 2 cats, and 1 fox suspected of developing vaccine-induced rabies were shown to have reactivity patterns that were identical to the vaccine administered. Reactivity patterns for 20 rabies isolates from human beings, wild animals, or domestic animals with no history of recent vaccination with modified-live virus rabies vaccine were different from those obtained for vaccines.     

Quantitation of histochemical staining of salivary gland mucin using image analysis in cats and dogs

Two different, computer-based, image analysis methods were employed to complement subjective assessment of patterns of mucin staining (by periodic acid Schiff/alcian blue, aldehyde fuchsin/alcian blue and potassium hydroxide-alcian blue/phenylhydrazine hydrocholoride) in digitised images of sections of major and minor salivary glands from cats and dogs. Image analysis based on red, green and blue (RGB) staining was not suitable for quantitation of histochemical staining of mucin in salivary glands. Image analysis based on hue, saturation and lightness (HSL) allowed quantitative assessment of staining by different stain components and of mixed staining, and enabled comparison of staining of different glands in dogs and cats. Quantitative analysis based on HSL allowed differentiation of differences in staining patterns of major and minor cat and dog salivary glands, and between the species; such differences would have been impossible to distinguish on subjective grounds alone. Quantitative assessment of normal salivary gland histochemistry allows comparison with staining patterns in disease.     

成都市区犬猫狂犬病病毒和弓形虫感染的调查

为了解四川省成都市区家养犬、猫狂犬病病毒及弓形虫的感染情况,采用商品化狂犬病病毒和弓形虫抗原快速检测试纸卡,对2010年8～10月间来自成都市区的103份家养犬以及75份家猫的唾液和血液样品进行检测;同时采用文献报道的PCR方法对家养犬血液样品中的弓形虫核酸进行检测。结果显示,103份家养犬唾液样品狂犬病病毒抗原均为阴性,而75份家猫唾液样品中,检出阳性样品5份(阳性率6.7%),可疑样品7份;103份家养犬血液样品弓形虫抗原阳性样品33份(32.0%),可疑样品22份,75份家猫血液样品中,检出阳性样品2份(阳性率2.7%),可疑样品3份。弓形虫核酸PCR检测结果显示,96份家养犬血液样品弓形虫核酸阳性样品57份(阳性率59.4%),与弓形虫抗原阳性和可疑样品总和所占比例基本一致(53.4%)。提示应重视源于家猫的狂犬病病毒和家养犬弓形虫对人的威胁。     

Adsorption of Wild Rabies Virus and Neurotropic-Adapted Viruses by Activated Attapulgite

The adsorption by attapulgite (a naturally occurring clay) of wild rabies virus in naturally rabid dogs was investigated. Attapulgite selectively absorbed rabies virus in the brain tissue of naturally rabid dogs and rejected rabies virus in extracts from the submaxillary salivary glands of the rabid dogs. Attapulgite rejected the neurotropic-adapted viruses lymphocytic choriomeningitis, Eastern equine and Western equine encephalitis, Japanese B encephalitis, and St. Louis encephalitis in the brain tissue of suckling mice.     

Immunofluorescence Study of Wild Rabies Virus and Antibody

A concentrate of wild rabies antibody was prepared from hyperimmune serums of three dogs refractory to wild rabies. The animals resisted repeated intramuscular injections of large doses of wild rabies virus in emulsions of whole brain, in emulsions of submaxillary salivary glands, and in emulsified mixtures of brain and submaxillary glands taken from naturally rabid dogs.

The antibody was conjugated with fluorochrome and then absorbed by a procedure that gave “cell-free” working solutions of fluorescent antibody. The procedure entailed parallel absorption steps with minced pathological canine submaxillary glands from (1) naturally rabid dogs (these glands contained specific, undegraded, natural antigens of live wild rabies virus plus nonspecific substances and antigens) and (2) nonrabid dogs from a rabies endemic region (these glands contained nonspecific substances and antigens).

Extracts from submaxillary glands of the three naturally rabid dogs and one nonrabid dog were stained with a cell-free solution of the fluorescent antibody. The glands of the rabid dogs contained fluorescent aggregates of intense green spherical and filamentous particles. When nonfluorescent canine hyperimmune serum was incubated with rabies-containing submaxillary extract, the rabies antigens were quenched. When nonfluorescent equine fixed virus antiserum was incubated with such extracts, the aggregates still retained bright fluorescence.

     

野生动物用狂犬病口服疫苗复合免疫佐剂与诱饵的筛选及其对免疫效果的影响

本研究旨在筛选最佳的复合佐剂和口服疫苗诱饵组方,并将筛选出的复合佐剂口服免疫试验小鼠,检测免疫小鼠血清IgG和肠道IgA抗体效价。采用牛肉粉、鸡肉粉、鱼肉粉、奶粉、血粉为主要原料,以细菌脂多糖、氢氧化锌、枸杞多糖、甘露低聚糖为疫苗免疫佐剂,通过正交试验方法将复合免疫佐剂和口服疫苗诱饵与狂犬病SRV₉病毒混匀后口服免疫试验小鼠,并对免疫动物血清IgG抗体效价进行检测。优化的复合佐剂组方是细菌脂多糖100 μg、氢氧化锌0.8 mg、枸杞多糖50 mg、甘露低聚糖10 mg,免疫小鼠血清IgG抗体含量为3.24 mg/L,肠道IgA抗体含量为1.56 ng/mL;在疫苗诱饵的投喂试验中,狼、狐狸、犬、猫对鱼肉味诱饵的采食率最高,其次是血味和奶味诱饵;诱饵与狂犬病SRV₉病毒混匀后口服免疫小鼠28 d后,血清IgG平均值为1.20 U/L。本试验筛选出口服疫苗复合免疫佐剂,经筛选制备的鱼肉味诱饵具备高采食性,适合野生食肉类动物口服疫苗的制备。可增加狂犬病SRV₉病毒的免疫效应,为进一步研发重组减毒狂犬病口服疫苗提供了必要的技术支撑。     

Experimental rabies in skunks: Immune response and salivary gland infection

Groups of striped skunks (Mephitis mephitis) were inoculated intramuscularly with graded doses of street rabies virus. At various intervals after inoculation, saliva and sera were tested for rabies virus and neutralizing antibodies, respectively. Skunks that developed rabies were killed in terminal stages of the disease and the following examinations were made: titers of virus and antibody in submandibular salivary glands and brain, extent of immunofluorescence in submandibular salivary glands, and histologic examination of various tissues.

Skunks that received inocula containing 4 × 10⁴ to 4 × 10⁵ mouse intracerebral lethal dose₅₀ (MICLD₅₀) had detectable serum neutralizing antibodies by 7–12 days postinoculation; however, most of the skunks that received lower doses (40 to 4 × 10³ MICLD₅₀) did not have detectable serum neutralizing antibodies until clinical signs began. In the salivary glands, slight and extensive immunofluorescence corresponded to high and low titers of tissue neutralizing antibody. Also low viral titers were associated with high tissue neutralizing antibody titers. There was a close correlation between viral titers in right and left submandibular salivary glands.

The results suggest that the immune response can impede the process of infection of the salivary glands resulting in lack of antigen or low amounts of antigen in this tissue. This could occur through interference with centrifugal neural transport of virus and/or neutralization of virus during transfer from neural elements to epithelial cells. Lack of infectious virus or low viral titers in salivary glands containing antigen and high levels of tissue neutralizing antibodies can be caused partly by postmortem virus neutralization (during viral titration).     

Seroconversion in captive African wild dogs (Lycaon pictus) following administration of a chicken head bait/SAG-2 oral rabies vaccine combination

This study determined the proportion of captive juvenile and adult African wild dogs (Lycaon pictus) that developed protective titres of rabies neutralising antibodies following ingestion of a chicken head bait/SAG-2 oral rabies vaccine combination. A single chicken head containing 1.8 ml of SAG-2 vaccine (10(8.0) TCID50/ml) in a plastic blister was fed to each of eight adult and three juvenile wild dogs. Bait ingestion resulted in a significant rise in serum neutralising antibody titres. Overall seroconversion rate was eight out of 11 (72.7%), and all the puppies and five out of eight (62.5%) adults showed potentially protective levels of antibodies on day 31. The mean post-vaccination neutralising antibody titre was within the range reported to be protective against challenge with virulent rabies virus in other species.