Epidemiology of hepatitis E virus in indoor-captive cynomolgus monkey colony

A serological survey of hepatitis E virus (HEV) antibody was conducted using 202 adult captive cynomolgus monkeys, who did not show any clinical signs of acute hepatitis. Out of these, 44 monkeys were sero-positive for anti-HEV IgG and all monkeys were negative for anti-HEV IgM. All positive monkeys came from either Vietnam or China, but none from the Philippines, Indonesia, or our facility. Selected 12 monkeys out of positive monkeys from Vietnam, including 9 positive and 3 negative, revealed mostly within the reference ranges for alanine aminotransferase (ALT) and asparatate aminotransferase (AST) by serum biochemistries. Their titers of anti-HEV IgG did not correlate with the concentrations of ALT and AST. Moreover, HEV-RNA could not be detected from any fecal specimens of the 12 monkeys. Thus, monkeys with anti-HEV IgG sero-positive did not seem to be source of the HEV-pollution, because 1) sero-positive monkeys did not excrete HEV-RNA from their feces, and 2) monkeys from the Philippines and Indonesia have remained to be sero-negative for anti-HEV IgG, even if the monkeys were kept in same animal room of our facility. From these results, it could be inferred that primary infection of HEV occurred in the exported countries, but not in our colony. The contamination of HEV in indoor-captive monkeys could be prevented by precise quarantine tests, including ELISA for detecting anti-HEV and RT-PCR for HEV RNA.     

The Incidence of Lymphoplasmacytic Gastritis in the Fundus and Antrum of Cynomolgus Monkey ( Macaca fascicularis ) Stomachs

Lymphoplasmacytic gastritis is a concern for toxicological pathologists reading preclinical, non-human primate toxicity studies because subtle gastric changes which could be treatment-related may be masked and gastritis lesions may be confused with treatment-related effects and thus a gastric finding may be incorrectly assigned as a treatment-related lesion. This paper discusses the incidence of lymphoplasmacytic gastritis in cynomolgus monkeys at a contract research organization. The incidence of lymphoplasmacytic gastritis in the fundus and antrum of control cynomolgus monkeys on 18 non-gastric compound studies, was scored. The average fundus score ranged from 0.3 to 1.5 and the average antral score ranged from 0.9 to 3.5 in the cynomolgus monkey stomachs examined. The number of affected control animals in a study ranged from 0 to 5 control animals. No correlation between the route of vehicle administration and the severity or incidence of the lesions was noted. The percentage incidence of affected animals ranged from 0 to 100%. An increased incidence lymphoplasmatic gastritis from 2000 to 2004 was noted. The implications of lymphoplasmacytic gastritis in cynomolgus monkeys used for acute toxicity studies are discussed.     

An outbreak of melioidosis in imported primates in Britain.

An outbreak of melioidosis, a bacterial infection caused by Pseudomonas pseudomallei, was identified in a batch of feral cynomolgus monkeys (Macaca fascicularis) imported to Britain from the Philippines. Thirteen confirmed or possible cases occurred among a batch of 50 animals. Subsequent investigations revealed that the infection was uncommon among imported primates from a variety of sources, although three other cases were identified in monkeys imported from Indonesia. The majority of the affected monkeys had splenic abscesses, and hepatic abscesses and infections of the soft tissues and skin were also frequently observed. Most of the infected animals had no clinical signs despite extensive abscesses, and the presence of infection was only suspected when they were shown to have serum antibodies to P pseudomallei by an enzyme-linked immunosorbent assay. Although there was no evidence of cross infection of other animals or human handlers, this outbreak is a reminder of the dangers of working with wild-caught primates and the potential for the establishment of environmental foci of melioidosis.     

Malaria in cynomolgus monkeys used in toxicity studies in Japan

Plasmodium spp. protozoa cause malaria and are known to infect humans and a variety of animal species including macaque monkeys. Here we report both our experience with malaria recrudescence in cynomolgus monkeys (Macaca fascicularis) in a toxicity study and the results of a survey on Plasmodium infection in cynomolgus monkeys imported to Japan for laboratory use. A cynomolgus monkey from the toxicity study presented with severe anemia and Plasmodium protozoa in erythrocytes on a thin blood smear and was subsequently diagnosed with symptomatic malaria. In this animal, congestion and accumulation of hemozoin (malaria pigment) in macrophages were noted in the enlarged and darkly discolored spleen. As a follow-up for the experience, spleen sections from 800 cynomolgus monkeys in toxicity studies conducted between 2003 and 2013 were retrospectively examined for hemozoin deposition as a marker of Plasmodium infection. The origin of the animals included Cambodia, China, Indonesia, and Vietnam. Hemozoin deposition was confirmed in 44% of all examined monkeys. Monkeys from Indonesia showed the highest incidence of hemozoin deposition (approx. 80%). A high prevalence of Plasmodium infection in laboratory monkeys was also confirmed with polymerase chain reaction (PCR) by using Plasmodium genus-specific primers. Although Japan is not a country with endemic malaria, it is important to be aware of the prevalence and potential impact of background infection with Plasmodium spp. and recrudescence of symptomatic malaria in imported laboratory monkeys on pharmaceutical toxicity studies.     

Helicobacter pylori-induced gastritis in experimentally infected conventional piglets.

A conventional nonmutant animal that could be experimentally infected with Helicobacter pylori isolates would be a useful animal model for human H. pylori-associated gastritis. Gnotobiotic and barrier-born pigs are susceptible to H. pylori infection, but attempts to infect conventional pigs with this bacterium have been unsuccessful. In the present study, a litter of eight 20-day-old crossbreed piglets were purchased from a commercial farm. Six of them were orally challenged two to five times at different ages, between 29 and 49 days, with doses of H. pylori inoculum containing approximately 10(9) bacterial cells. Two animals served as controls. The inoculation program began 2 days postweaning when the piglets were 29 days of age. Prior to every inoculation, the piglets were fasted and pretreated with cimetidine, and prior to the first and second inoculation each piglet also was pretreated with dexamethasone. The challenged piglets were euthanasized between 36 and 76 days of age. H. pylori colonized all six inoculated piglets. The pathology of the experimentally induced gastritis was examined macroscopically and by light and electron microscopy. H. pylori induced a severe lymphocytic gastritis in the conventional piglets and reproduced the large majority of the pathologic features of the human disease. Therefore, the conventional piglet represents a promising new model for study of the various pathogenic mechanisms involved in the development of lesions of the human H. pylori-associated gastritis.     

Investigation of Helicobacter pylori in raw sheep milk samples

It is known that Helicobacter pylori is a cause of chronic gastritis and peptic ulcer disease in humans. However the origin and transmission of this bacterium has not been clearly explained. One of the suggested theories is transmission via raw milk from animals to human beings. In this study, the presence of H. pylori was investigated in sheep milk that is commonly consumed as human food. For this purpose, a total of 440 raw sheep milk samples collected from the Burdur region of Turkey were examined by specific cultural procedures, however, H. pylori was not isolated in any sample.     

Histopathology of incidental findings in cynomolgus monkeys ( macaca fascicularis ) used in toxicity studies

The purpose of our publication is to widely communicate pictures of spontaneous findings occurring in cynomolgus monkeys. Focal lymphoplasmacytic infiltration is commonly seen in the general organs. The frequency and severity of these lesions may be influenced by the administration of drugs with an effect on the immune system. Lymphoplasmacytic infiltration in the lamina propria of the stomach is also frequently seen in cynomolgus monkeys, and it is caused mainly by a Helicobacter pylori infection. Various degrees of brown pigments are observed in various organs, and it is possible to distinguish the material of the pigments by its morphological features and site. A focal/segmental glomerular lesion is occasionally seen in a section of the kidney, and the minimal lesion has no influence on the urinalysis. We showed the common glomerular lesions in HE-stained sections, as well as in PAM- or PAS-stained sections, for understanding the details. Young and pubertal monkeys are usually used in toxicity studies; therefore, understanding various maturation stages of the genital system is important. In particular, the female genital system needs to be understood in the morphology, because their cyclic changes are different from other laboratory animals. Thus, we present the normal features of the cyclic changes of the female genital organs. Furthermore, we provide more information on spontaneous findings in cynomolgus monkeys for exact diagnoses in toxicity studies.     

Genome sequence of Helicobacter suis supports its role in gastric pathology

ABSTRACT: Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present.     

Seroprevalence of BV (Macacine herpesvirus 1) in bred cynomolgus monkeys in Cambodia

The seroprevalence of B Virus (BV, Macacine herpesvirus 1) in bred cynomolgus monkeys (Macaca fascicularis) in Cambodia was investigated. Blood samples were collected by venous puncture between 2007 and 2011, and a commercially available enzyme-linked immunosorbent assay (ELISA) kit was used to detect IgG antibodies against BV. A total of 1710 blood samples were examined, and the overall prevalence of BV antibodies was 33.92% (580/1710). The young adult (2-6 years) monkeys had a seroprevalence of 30.57% (497/1626), and the older (>7 years) monkeys had a seroprevalence of 98.81% (83/84). The present investigation revealed that the prevalence of BV antibodies in bred cynomolgus monkeys was highly correlated with their age, with higher prevalence in older monkeys in Cambodia.     

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.     

Did transmission of Helicobacter pylori from humans cause a disease outbreak in a colony of Stripe-faced Dunnarts (Sminthopsis macroura)?

ABSTRACT: Since the discovery that Helicobacter pylori causes a range of pathologies in the stomachs of infected humans, it has become apparent that Helicobacters are found in a diverse range of animal species where they are frequently associated with disease. In 2003 and 2004, there were two outbreaks of increased mortality associated with gastric bleeding and weight-loss in a captive colony of the Australian marsupial, the Stripe-faced Dunnart (Sminthopsis macroura). The presence of gastric pathology led to an investigation of potential Helicobacter pathogenesis in these animals. Histological examination revealed the presence of gastritis, and PCR analysis confirmed the presence of Helicobacter infection in the stomachs of these marsupials. Surprisingly, sequencing of 16S rRNA from these bacteria identified the species as H. pylori and PCR confirmed the strain to be positive for the important pathogenesis factor, cagA. We therefore describe, for the first time, an apparent reverse zoonotic infection of Stripe-faced Dunnarts with H. pylori. Already prone to pathological effects of stress (as experienced during breeding season), concomitant H. pylori infection appears to be a possible essential but not sufficient co-factor in prototypic gastric bleeding and weight loss in these marsupials. The Stripe-faced Dunnart could represent a new model for investigating Helicobacter-driven gastric pathology. Infections from their human handlers, specifically of H. pylori, may be a potential risk to captive colonies of marsupials.     

Age-dependent seroprevalence of antibodies against a Helicobacter pylori-like organism and Helicobacter pylori in commercially reared swine

OBJECTIVE: To determine the prevalence of antibodies against a swine-origin Helicobacter pylori-like organism (HPLO) and H pylori in conventionally reared swine. ANIMALS: 640 conventionally reared swine of various ages from 16 high-health farms in Canada, 20 sows from Ohio, and 35 gnotobiotic swine. PROCEDURES: Blood was collected from the cranial vena cava. Sera were collected and tested via ELISA for antibodies against antigen prepared from a swine-origin HPLO and human H pylori strain 26695. RESULTS: Antibodies reactive with a swine HPLO, H pylori, or both were detected in 483 of 640 swine from all 16 farms in western Canada. Seroprevalence varied with age and was low (5.6%) in suckling ( 4 weeks old to adulthood. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that colonization by a swine-origin HPLO, H pylori, or both and resultant seroconversion, like that of H pylori infection in humans, were common in commercial swine operations. Furthermore, data indicated that gastric infection was acquired at an early age. The relationships to gastric colonization by HPLOs and clinical manifestations of disease such as gastritis and gastroesophageal ulceration remain to be determined.     

Pathology, diagnosis and epidemiology of the rodent Helicobacter infection

Since the first isolation of Helicobacter pylori from humans in 1983, 18 Helicobacter species have been identified during the last decade in domestic and laboratory animals. Several Helicobacter species have been isolated from the gastrointestinal tracts of various mammalian species and birds. Helicobacter hepaticus, H. muridarum, H. bilis, H. rodentium and Flexispira rappini have been isolated from mice. Among these species, only H. hepaticos has been clearly recognized as a pathogen. Indeed, it displays the pathogenic potential to elicit hepatitis in several strains of mice; moreover in A/JCr mice, it is strongly associated with hepatic cancer. Among the five murine helicobacter species, apart from H. hepaticus, F. rappini has not been found associated with lesions, H. muridarum has been observed in gastric glands of mice with chronic gastritis, and H. bilis has been reported in the liver of mice with chronic hepatitis. When associated with H. rodentium, H. bilis is able to induce diarrhea in SCID mice. In no case has pathogenicity of a single species been clearly proven. In rats, H. trogontum and H. muridarum have been isolated from the intestine, without any information concerning their respective pathogenicity. H. cinaedi and H. cholecystus have been identified from the intestine and the gallbladders of hamsters, respectively. The diagnosis of Helicobacter species by polymerase chain reaction (PCR) is a rapid, specific and sensitive technique. One of the most promising diagnostic techniques of these infections seems to be the PCR detection of Helicobacter sp. from feces based on the 16S rRNA sequences, then a restriction enzyme analysis to identify the actual species. Several drug regimens have also been evaluated to eradicate H. hepaticus from mice. Helicobacter infections, particularly H. hepaticus and H. bilis, seem to be widespread in laboratory mouse colonies and have also been detected from commercial breeders.     

Gastritis associated with Helicobacter-like organisms in baboons

Subclinical gastritis was observed in 10 of 10 baboons (Papio spp.) from a toxicity study in a research facility. The lesions were similar in xenobiotic-treated and control animals, suggesting a spontaneous rather than chemical-induced disease. Histologic examination revealed lymphoplasmacytic gastritis in the antral mucosa. The fundic mucosa contained minor, scattered aggregates of lymphocytes and plasma cells. A Warthin-Starry silver stain and ultrastructural examination revealed numerous spiral-shaped bacteria morphologically resembling Helicobacter pylori in antral glands and numerous spiral-shaped bacteria morphologically consistent with H. heilmannii-like organisms in fundic glands. Polymerase chain reaction assay of paraffin-embedded antral and fundic tissue using primers for the urease gene and 16S ribosomal ribonucleic acid gene amplified deoxyribonucleic acid fragments with a high degree of sequence homology for H. pylori and H. heilmannii. This is the first report of gastritis associated with Helicobacter-like organisms in baboons.     

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte,Liguria e Valle d’Aosta,Italy

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016–2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.     

Isolation of Helicobacter mustelae from ferrets in New Zealand

AIMS: The bacterial genus Helicobacter contains over 20 species, including the human gastric pathogen H. pylori, and the mustelid-specific H. mustelae. A previous study in this country failed to isolate H. mustelae from a captive breeding colony of ferrets. We sought to confirm whether or not H. mustelae was present in this country. METHODS: A combination of bacterial culture, phenotypic testing and molecular techniques were used to isolate and identify gastric bacteria from captive and wild populations of ferrets in the New Zealand North Island. RESULTS: Bacteria were isolated from captive and wild ferrets which were phylogenetically identical to the type strain of H. mustelae. A mild to moderate gastritis was seen in five of six animals examined, and an antibody response to H. mustelae proteins was demonstrated. CONCLUSIONS: Helicobacter mustelae is not exotic to New Zealand, but is present in two populations of ferrets tested in the North Island.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Molecular characteristics of canine parainfluenza viruses type 5 (CPIV-5) isolated in Korea

Three canine parainfluenza viruses type 5 (CPIV-5) were isolated from lung tissues of 3 Korean dogs with mild pneumonia between 2008 and 2009. The isolates were fully sequenced and compared with published reference sequences. The size of the genome was 15 246 nucleotides long and no remarkable differences were found when compared with previously published reference sequences. In phylogenetic analysis based on the F and P genes, parainfluenza virus 5 (PIV-5) strains were divided into at least 3 subgroups. Three CPIV-5 strains were clustered with CPIV-5 T1, H22 and 78524 strains. All PIV-5 strains were independent of the host species, geographical distribution, and the isolated period.     

Prevalence of antibodies to five selected zoonosis agents in monkeys.

The prevalence of antibodies against 5 zoonosis agents was determined in serum samples of 443 breeding monkeys. Of the monkeys, 296 were bred or kept for a long time at R institute, and the remaining 147 were newly imported from the Philippines and kept at S institute for quarantine. Antibodies to simian virus 40 were highly prevalent at 89.1% among monkeys of R institute, whereas no antibody could be detected in those of S institute. Antibodies to Chlamydia psittaci and Yersinia pseudotuberculosis were detected in 14.4 and 11.6% at R institute, and in 9.0 and 3.5% at S institute, respectively, evidencing a significant difference (P less than 0.05) between those of the two institutes for both agents. Antibodies to Toxoplasma gondii and Leptospira interrogans were found in 3.6 and 2.9% of the overall, respectively, showing no difference in positive rates in relation to breeding place. Even in cases positive to the latter 4 zoonosis agents, the antibody titers were low. The results obtained suggest that all zoonoses tested do not seem to be so serious diseases among monkeys at the present time except simian virus 40 infection, which is highly prevalent among breeding monkeys in Japan.