Altered cannabinoid receptor expression in pancreatic islets in experimental model of uraemia.

BACKGROUND: Uraemia leads to a number of metabolic and hormonal disorders including defective carbohydrate metabolism. Endocannabinoids exert their effect on insulin and glucagon secretion via activation of specific receptors named CB1 and CB2. For this reason and the absence of reports on location and immunoreactivity of CB1, CB2 receptors compared to immunoreactivity of insulin- and glucagon-secreting cells in experimental uraemia, the author decided to investigate this issue. The aim of the present study was the immunohistochemical localisation and evaluation of cannabinoid receptors (CB1, CB2), insulin and glucagon in the pancreatic islets of uraemic rats. MATERIALS AND METHODS: Fragments of the rat's pancreas were collected 28 days after surgical resection of one kidney and removal of 70% of the other kidney cortex. Paraffin-embedded sections were stained with haematoxylin-eosin and immunohistochemical reactions were performed with the use of a specific antibody against CB1-, CB2-receptors, insulin and glucagon. RESULTS: It was revealed the decreased immunoreactivity of the CB1 receptor and higher intensity of the immunohistochemical reaction against CB2 receptor as compared to the value in the control animals. Significantly higher immunoreactivity of glucagon-positive cells and weaker immunoreactivity of insulin-positive cells were observed in pancreatic islets of uraemic rats. CONCLUSIONS: The obtained results indicate the involvement of cannabinoid receptors in the pathomechanism of carbohydrate metabolism disorders, associated with abnormal secretion of hormones by the α and ß cells in uraemia.

Clinical management and humoral immune responses to rabies post-exposure prophylaxis among three patients who received solid organs from a donor with rabies.

BACKGROUND: The rabies virus causes a fatal encephalitis and can be transmitted through organ transplantation. In 2013, a man developed rabies 18 months after receiving a kidney from a donor with rabies, who was not known to have been infected when the organs were procured. Three additional persons who received organs from the same donor (liver, kidney, heart), all of whom were not vaccinated for rabies before transplantation, received rabies post-exposure prophylaxis (PEP) with rabies immune globulin and 5 doses of rabies vaccine as soon as the diagnosis of rabies was made in the donor (18 months after their transplant surgeries). We describe their clinical management. METHODS: As the 3 recipients were all on immunosuppressive medications, post-vaccination serologic testing was performed using the rapid fluorescent focus inhibition test to measure rabies virus neutralizing antibodies (RVNAs). An acceptable antibody response to administration of rabies vaccine was defined as detection of RVNAs at a concentration ≥0.1 IU/mL from a serum specimen collected ≥7 days after the fifth vaccine dose. RESULTS: All 3 recipients demonstrated an acceptable antibody response despite their immunosuppressed states. More than 36 months have passed since their transplant surgeries, and all 3 recipients have no evidence of rabies. CONCLUSIONS: The survival of 3 previously unvaccinated recipients of solid organs from a donor with rabies is unexpected. Although the precise factors that led to their survival remain unclear, our data suggest that PEP can possibly enhance transplant safety in settings in which donors are retrospectively diagnosed with rabies.

High prevalence of antibodies against canine adenovirus (CAV) type 2 in domestic dog populations in South Africa precludes the use of CAV-based recombinant rabies vaccines.

Rabies in dogs can be controlled through mass vaccination. Oral vaccination of domestic dogs would be useful in the developing world, where greater vaccination coverage is needed especially in inaccessible areas or places with large numbers of free-roaming dogs. From this perspective, recent research has focused on development of new recombinant vaccines that can be administered orally in a bait to be used as adjunct for parenteral vaccination. One such candidate, a recombinant canine adenovirus type 2 vaccine expressing the rabies virus glycoprotein (CAV2-RG), is considered a promising option for dogs, given host specificity and safety. To assess the potential use of this vaccine in domestic dog populations, we investigated the prevalence of antibodies against canine adenovirus type 2 in South African dogs. Blood was collected from 241 dogs from the Gauteng and KwaZulu-Natal provinces. Sampled dogs had not previously been vaccinated against canine adenovirus type 1 (CAV1) or canine adenovirus type 2 (CAV2). Animals from both provinces had a high percentage of seropositivity (45% and 62%), suggesting that CAV2 circulates extensively among domestic dog populations in South Africa. Given this finding, we evaluated the effect of pre-existing CAV-specific antibodies on the efficacy of the CAV2-RG vaccine delivered via the oral route in dogs. Purpose-bred Beagle dogs, which received prior vaccination against canine parvovirus, canine distemper virus and CAV, were immunized by oral administration of CAV2-RG. After rabies virus (RABV) infection all animals, except one vaccinated dog, developed rabies. This study demonstrated that pre-existing antibodies against CAV, such as naturally occurs in South African dogs, inhibits the development of neutralizing antibodies against RABV when immunized with a CAV-based rabies recombinant vaccine.

Enhancing comparative rabies DNA vaccine effectiveness through glycoprotein gene modifications.

Enhancing DNA vaccine effectiveness remains a challenge, especially if the desired goal is immunization efficacy after a single dose. The glycoprotein gene from the rabies virus Evelyn-Rokitnicki-Abelseth (ERA) strain was modified by mutation at amino acid residue 333 from arginine to glutamine. The modified and original unmodified glycoprotein genes were cloned separately and developed as DNA vaccines for immunization in mice. The intramuscular (IM) route using a single dose (100 microg) of a modified DNA vaccine showed virus neutralizing antibody induction by d30, and 80% of the mice survived a challenge in which 100% of unvaccinated controls succumbed. Similar results were obtained using a single dose (10 microg) by the intradermal (ID) route with one-tenth amount of the DNA administered. Administration of single dose of DNA vaccine with unmodified G did not result in the production of detectable levels of virus neutralizing antibody by d30. The results of the IM and the ID routes of administration were statistically significant (P<0.01). Based on these preliminary results, a modified glycoprotein gene from the ERA rabies virus strain may be an ideal candidate for DNA vaccine efficacy enhancement.

Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia.

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

A comparison of DNA vaccines for the rabies-related virus, Mokola.

Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge.

The prevalence of interstitial nephritis and leptospirosis in 283 raccoons (Procyon lotor) from 5 different sites in the United States.

A retrospective histopathological study was carried out on tissues of 283 raccoons from 5 different geographical locations for presence of interstitial nephritis and renal leptospirosis. Results of this study indicate that although interstitial nephritis was common in raccoons from all locations, the presence of renal leptospiral spirochetes was not.

Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination.

This study investigated the safety, efficacy, and clearance of SAG-2, an attentuated rabies virus, after oral vaccination in dogs. Nineteen dogs consumed baits containing lyophilized vaccine, but residual SAG-2 virus was recovered in only one of 57 oral swabs, collected one hour post-vaccination. Seven vaccinates were euthanized between 24 and 96 h after consuming a bait. Rabies virus RNA was detected in tonsils from all seven dogs by nested RT-PCR, with primers to the viral glycoprotein. Genomic, sense-transcripts, and m-RNAs were detected in five of seven tonsil samples using primers to the rabies virus nucleoprotein gene, as well as in four of seven samples from the buccal mucosa and one of seven from the tongue. Rabies virus antigen was detected in all tonsils by an immunohistochemistry test, confirming the RT-PCR results. In addition, virus was isolated from one tonsil sample collected at 96 h, providing supportive evidence of viral replication. Ten of 12 (83%) of the vaccinated dogs demonstrated an anamnestic response, with viral neutralizing antibody titers (> or =0.5 IU/ml), after rabies virus challenge. These ten dogs survived, whereas all control dogs succumbed to rabies. Attenuated rabies viruses, such as SAG-2, replicate in local tissues of the oral cavity and can be cleared relatively quickly, without viral excretion, leading to protective immunity against the disease.

Experimental utility of rabies virus-neutralizing human monoclonal antibodies in post-exposure prophylaxis.

Rabies immune globulin (RIG) is essential for post-exposure prophylaxis but is expensive and not widely available. Rabies virus-neutralizing human monoclonal antibodies (Mabs) were evaluated in vitro and in a Syrian hamster model as a potential future alternative. Seven Mabs neutralized representative rabies virus variants. However, a European bat lyssavirus was not neutralized by either Mabs or RIG. Moreover, Duvenhage virus was neutralized by RIG, but not by Mabs, and Lagos bat and Mokola viruses were neutralized by one Mab but not by RIG. In hamsters, one Mab resulted in protection that was comparable to human RIG. These results suggest that Mabs may provide a promising alternative to RIG.

A comparative study of the fluorescent antibody test for rabies diagnosis in fresh and formalin-fixed brain tissue specimens.

Many diagnostic methods have been used to detect rabies virus antigen. The preferred method for routine diagnosis of rabies in fresh or frozen brain tissues is the fluorescent antibody test (FAT). In this study, the FAT was used to evaluate the rabies status of fresh/frozen brain specimens from more than 800 rabies-suspected cases, in more than 14 different species of animals. A comparable brain specimen from each case was fixed in 10% buffered formalin and examined by the FAT. The evaluation of rabies status between fresh and formalin-fixed tissues was in agreement in more than 99.8% of the cases. When fresh tissue is not available for testing, these results validate the use of this procedure for routine diagnosis of rabies in formalin-fixed brain tissues.

The incurable wound revisited: progress in human rabies prevention?

Rabies is the most important viral zoonosis from a global perspective. Modern human postexposure prophylaxis consists of potent vaccines and local infiltration of rabies immune globulins (RIGs), but the latter biologicals are not widely available or affordable. Monoclonal antibodies (Mabs) offer several theoretical advantages over RIGs. To this end, several human and equine RIGS, alone or in combination with vaccine, were investigated for postexposure efficacy in a Syrian hamster model, compared with a single neutralizing murine Mab. Preliminary results suggest that: (1) animal models continue to provide utility as human surrogates in the demonstration of product efficacy against rabies; (2) RIG preparations differ substantially in experimental effectiveness and clearance; and (3) relevant alternatives, such as Mabs, should be pursued for future improvements to human rabies prevention.

Serologic survey for selected infectious disease agents in swift and kit foxes from the western United States.

A serologic survey of swift fox (Vulpes velox) and kit fox (V. macrotis) from the western USA was conducted for 12 infectious diseases. Samples from swift fox were collected between 1987 and 1992 from Colorado (n = 44), Kansas (n = 10), and Wyoming (n = 9). Samples from kit fox were collected in California (n = 86), New Mexico (n = 18), Utah (n = 9), and Arizona (n = 6). Overall antibody prevalence rates were 33 of 110 (30%) for canine parvovirus (CPV), 9 of 72 (13%) for canine distemper virus (CDV), 23 of 117 (20%) for vesicular stomatitis New Jersey, 16 of 117 (14%) for vesicular stomatitis Indiana, six of 117 (5%) for Cache Valley virus, five of 117 (4%) for Jamestown Canyon virus, one of 97 (1%) for rabies virus, one of 117 (1%) for Colorado tick fever virus, and one of 117 (1%) for western equine encephalitis virus. In addition, antibodies were not found to Yersinia pestis, Francisella tularensis, and Borrelia burgdorferi in serum from 25 Colorado swift fox. Adult swift fox from Colorado had serologic evidence of exposure to CPV more often than juveniles. No juvenile swift fox from Colorado had serum antibodies to CDV. There were season-specific differences in serum antibody prevalence for CPV for swift fox from Colorado. No viruses were isolated from ectoparasites or fox from Colorado.

Rabies in an American bison from North Dakota.

In North Dakota (USA) during April 1998, a ranched female bison (Bison bison) was found dead. At gross necropsy, there was profound hair loss and consolidated lung lobes. Intracytoplasmic neuronal inclusions suggestive of Negri bodies were observed in the brain stem and hippocampus, and a diagnosis of rabies was confirmed by the fluorescent antibody test. Antigenic typing demonstrated the occurrence of a rabies virus variant associated with skunks from the upper midwestern USA. This case of a rabid bison was one of only four such instances recorded from the USA over the past 40 yr, and is the first case report of rabies in a bison that reports clinical, pathologic, and antigenic findings. Although rabies in bison is rare, veterinarians and wildlife managers that work closely with such non-traditional species are reminded of the dangers that zoonoses such as rabies present.

Laboratory investigation of human deaths from vampire bat rabies in Peru.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.

Viral excretion in domestic ferrets (Mustela putorius furo) inoculated with a raccoon rabies isolate.

OBJECTIVE: To determine susceptibility, incubation and morbidity periods, clinical signs of infection, serologic response, and excretion of virus in domestic ferrets inoculated with rabies virus of raccoon origin. ANIMALS: 54 domestic ferrets. PROCEDURE: 5 groups of ferrets were inoculated IM with the rabies virus. Oral cavity swab specimens and saliva were obtained for virus isolation. Blood was obtained for virus-neutralizing antibody determination. If clinical signs were severe, ferrets were euthanatized immediately. Salivary gland and brain tissue was collected for virus isolation and rabies diagnosis, respectively. RESULTS: Of 51 inoculated ferrets, 19 (37%) were euthanatized with clinical signs of rabies. Mean incubation period was 28 days (range, 17 to 63 days). Clinical signs included ataxia, cachexia, inactivity, paresis, paraparesis, bladder atony, tremors, hypothermia, lethargy, constipation, paralysis, and anorexia. Two rabid ferrets manifested aggressive behavior. Mean morbidity period was 4 to 5 days (range, 1 to 8 days). Virus antigen was detected in brain tissue from all rabid ferrets (n = 19). Two rabid ferrets had detectable virus-neutralizing antibody. Of 32 ferrets that survived, only 1 seroconverted; survivors remained clinically normal throughout the observation period. Rabies virus was isolated from salivary glands of 12 of 19 (63%) rabid ferrets, and 9 (47%) shed virus in saliva. Initiation of virus excretion ranged from 2 days before onset of illness to 6 days after onset. CONCLUSIONS AND CLINICAL RELEVANCE: Rabies should be considered in the differential diagnosis for ferrets that have acute onset of paralysis or behavioral changes and a condition that rapidly deteriorates despite intense medical intervention.

Prevention of the spread of rabies to wildlife by oral vaccination of raccoons in Massachusetts.

OBJECTIVE: To evaluate the use of bait containing rabies vaccine to create a barrier of rabies-vaccinated raccoons in Massachusetts and to determine the effectiveness of various bait distribution strategies in halting the spread of rabies. DESIGN: Prospective study. SAMPLE POPULATION: Free-ranging raccoons. PROCEDURE: Baits were distributed twice yearly in a 207-km2 (80-mi2) area in the vicinity of the Cape Cod Canal. Bait density and distribution strategy varied among 3 treatment areas. Raccoons were caught in live traps after bait distribution and anesthetized; blood samples were obtained to measure serum antibody titers to rabies virus. Vaccination rates were determined by the percentage of captured raccoons with antibody titers to rabies virus > or = 1:5. In addition, raccoons with clinical signs of illness inside the vaccination zone and adjacent areas were euthanatized and submitted for rabies testing. RESULTS: The percentage of vaccinated raccoons differed significantly among the following 3 areas with various bait densities: high-density area with uniform bait distribution (103 baits/km2 [267 baits/mi2]) = 37%; low-density area with additional targeted bait distribution (93 baits/km2 [240 baits/mi2]) = 67%; and, high-density area with additional targeted bait distribution (135 baits/km2 [350 baits/mi2]) = 77%. Nineteen animals with rabies (15 raccoons, 3 skunks, 1 cat) were reported in the area just outside of the vaccination zone, but only 1 raccoon with rabies was reported from inside the vaccination zone. CLINICAL IMPLICATIONS: In this suburban study area, an approximate vaccination rate of 63% was sufficient to halt the spread of rabies in free-ranging raccoons. Compared with uniform bait distribution, targeting raccoon habitats increased vaccination rates.

Efficacy of an oral vaccinia-rabies glycoprotein recombinant vaccine in controlling epidemic raccoon rabies in New Jersey.

A field trial to evaluate the efficacy of an oral vaccinia-rabies glycoprotein recombinant virus vaccine in controlling epidemic raccoon (Procyon lotor) rabies was conducted by distributing 180,816 doses (10(8.2)TCID50/ml) of vaccine in wax ampules within fish-meal polymer baits at a rate of 64 doses/km2/treatment throughout a 552 km2 area, forming an 18 km wide band across the northern Cape May Peninsula of New Jersey (USA). Vaccination treatments were conducted in the spring and fall between May 1992 and October 1994 from a helicopter along ecotones and from motor vehicles along roads. Vaccine-laden baits were removed by animals from tracking stations within 3 wk and 61% of the identifiable tracks were those of raccoons. Tetracycline incorporated in the baits as a biomarker was detected in 155 (73%) of the vaccination area raccoons following the fall 1993 and spring 1994 vaccinations. Eleven (61%) of the raccoons sampled in the same time period seroconverted (> or = 0.5 IU) in response to rabies virus glycoprotein. A raccoon diagnosed with rabies from the northern border of the vaccination area on 30 April 1993 provided the first evidence that the barrier was being challenged by the rabies epidemic. The prevalence of rabies in raccoons from the vaccination area for the first year (10%, n = 96) and second year (8%, n = 61) of challenge was reduced more than six-fold by vaccination compared to unvaccinated raccoons from northern adjacent surveillance areas during the corresponding first (65%, n = 189) and second years (53%, n = 43). Vaccination also effectively reduced by three-fold the rate at which the epidemic moved through the raccoon population (15 km/yr). The breach of the vaccination area resulted in a resumption of the high rate (43 km/yr) of epidemic movement and a significant nine-fold increase in rabies prevalence (77%, n = 47). The maximum linear movement (12.9 km) among five ear-tagged rabid raccoons in the study area was significantly greater than that of 19 normal radio-collared raccoons (2.58 km) in the area. These large movements of rabid raccoons, together with relocation of nuisance raccoons, spillover of raccoon rabies in skunks (Mephitis mephitis) and other species, insufficient funding and a decision to discontinue the program in 1994 (which could have resulted in insufficient population immunity among raccoons in the vaccination area) may have contributed to the eventual breach of the barrier.

First North American field release of a vaccinia-rabies glycoprotein recombinant virus.

Following nearly 10 yr of extensive laboratory evaluation, a vaccinia-rabies glycoprotein (V-RG) vaccine was the first recombinant virus to undergo limited North American field release on 20 August 1990. The free-ranging raccoon population on Parramore Island (Virginia, USA) was exposed to a high density (10 baits/ha) of vaccine-laden baits distributed on a 300 ha vaccination area. An annual total of 887 raccoons were live-trapped for sedation, physical examination and blood collection for rabies antibody determination; there was no evidence of adverse effects or lesions due to the vaccine. Age and sex distributions, mean body weights, and live-capture histories of raccoons from the vaccination and non-baited control areas were compared. There were no statistically significant differences in survivorship between the baited and non-baited areas, nor between rabies antibody-positive and antibody-negative raccoons from the vaccination area. There was no trend in field mortality that suggested an association with either tetracycline or sulfadimethoxine, used as biomakers, or with vaccine contact determined by antibody status. No gross or histopathologic lesions due to the vaccine were demonstrated among a subsample of live-trapped raccoons collected for gross necropsy, biomarker analysis, histopathologic examination, and V-RG virus isolation attempts. Recovery of V-RG virus was limited to the tonsils of two biomarker-positive, clinically healthy raccoons collected from the vaccination area for postmortem examination on days 2 and 4 following bait distribution. These data reinforce the extensive body of safety data on the V-RG virus and extend it to include field evaluation where vaccine is offered free-choice in abundance, in baits designed to attract free-ranging raccoons, in a relatively simple ecosystem.

Pathogenesis of experimentally induced rabies in domestic ferrets.

OBJECTIVE: To determine susceptibility, incubation and morbidity periods, clinical signs, serologic response, and excretion of virus in domestic ferrets inoculated with rabies virus. ANIMALS: 55 domestic ferrets. PROCEDURE: 5 groups of 10 ferrets were inoculated with rabies virus, IM, at doses of 10(5.5) to 10(1.5) median mouse intracerebral lethal dose. Ferrets were observed and behavior was recorded. Rectal temperature, body weight, and samples from the oral cavity and samples of saliva and blood were obtained. Virus isolation was attempted, using intracranial mouse inoculation and cell culture. Virus neutralizing antibodies were determined by rapid fluorescent focus inhibition test. Ferrets were euthanatized immediately if clinical signs were severe. Rabies was confirmed by direct immunofluorescent antibody test. RESULTS: Mean incubation period was 33 days (range, 16 to 96 days). Clinical signs included ascending paralysis, ataxia, cachexia, bladder atony, fever, hyperactivity, tremors, and paresthesia. Mean morbidity period was 4 to 5 days (range, 2 to 10 days). Virus antigen was detected in brain tissue from all clinically rabid ferrets. Ferrets given the highest viral dose were euthanatized and had VNA; ferrets receiving the next dilution also were euthanatized, but only 4 had seroconverted. Of 17 ferrets that survived, 5 seroconverted. Survivors remained clinically normal except for 1 that recovered with severe paralytic sequelae. Rabies virus was isolated from the salivary gland of 1 ferret that was euthanatized. CONCLUSIONS AND CLINICAL RELEVANCE: Rabies should be considered as a differential diagnosis in any ferret that has acute onset of paralysis or behavioral changes and a condition that rapidly deteriorates despite intense medical intervention.