Research Note: Antibiotic resistance and pathogenicity of geese-derived Riemerella anatipestifer isolated from eastern regions of Hebei Province, China.

Riemerella anatipestifer (R. anatipestifer) can cause serositis in multiple poultry species, resulting in significant losses. Although R. anatipestifer-caused infections in ducks have been well established, the literature about this disease in geese is rare. Here, we isolated and identified 56 strains of R. anatipestifer from the eastern regions of Hebei Province, China, and further determined their serotypes, antibiotic resistance, and pathogenicity. A total of 75 strains of causative bacteria were isolated from 70 sick geese with serositis. After Gram staining microscopy, PCR, and 16S rDNA sequence analysis, 56 isolates were identified as members of R. anatipestifer and 19 as Escherichia coli (E. coli). The results of serotyping showed that there were 4 serotypes prevalent in the isolate, including serotype 1 (37/56), serotype 2 (9/56), serotype 11 (8/56), and serotype 13 (2/56). The results of antibiotic susceptibility testing revealed that all 56 R. anatipestifer isolates showed varying degrees of multidrug resistance (MDR). A total of 10 antibiotic resistance genes (ARG) were determined in these isolates. Four isolates of different serotypes were selected for pathogenicity examination, and all were able to reproduce serositis-like symptoms in 15-day-old goslings, with neurological symptoms and a 100% mortality rate. Hemorrhagic congestion of the brain tissue, steatosis of the hepatocytes, and disorganization of some cardiac myofibers were observed in R. anatipestifer-infected geese. All these findings will contribute to our insights into the prevalence characteristics, antibiotic resistance profile, and pathogenicity of R. anatipestifer infection in geese in eastern Hebei Province and provide scientific guidance for the treatment and control of this disease.

Immunogenicity of live phoP gene deletion strain of Riemerella anatipestifer serotype 1.

Duck infectious serositis is an acute and infectious disease caused by Riemerella anatipestifer (R. anatipestifer) that leads to perihepatitis, pericarditis, meningitis, and airbag inflammation in ducks, which causes serious economic losses to the global duck industry. The phoP/phoR is a novel 2-component signal transduction system first reported in gram-negative bacteria, of which phoP acts as a global regulator and virulence factor. In this study, the phoP gene from the R. anatipestifer YM strain was knocked out using homologous recombination technology and replaced with the spectinomycin resistance gene (Spec). The virulence of the R. anatipestifer YMΔphoP strain was reduced by approximately 47,000 times compared to that of the wild-type R. anatipestifer YM strain. Ducks were immunized with live R. anatipestifer YMΔphoP strain by subcutaneous inoculation at a dose of 106 to 107 CFU (0.2 mL per duck) and challenged with the wild-type R. anatipestifer YM strain 14 days later. The protection rate in the immunized group was 100%. The growth characteristics of ducks in the immunized and negative control groups were normal, and the research demonstrated R. anatipestifer YMΔphoP strain have suitable immunogenicity and protective effects. Thus, the study findings suggest that the novel R. anatipestifer YMΔphoP strain may provide a candidate for the development of a gene deletion activated vaccine against duck infectious serositis.

Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis.

An emaciated subadult free-ranging California sea lion (Csl or Zalophus californianus) died following stranding with lesions similar to 11 other stranded animals characterized by chronic disseminated granulomatous inflammation with necrotizing steatitis and vasculitis, involving visceral adipose tissues in the thoracic and peritoneal cavities. Histologically, affected tissues had extensive accumulations of macrophages with perivascular lymphocytes, plasma cells, and fewer neutrophils. Using viral metagenomics on a mesenteric lymph node six mammalian viruses were identified consisting of novel parvovirus, polyomavirus, rotavirus, anellovirus, and previously described Csl adenovirus 1 and Csl bocavirus 4. The causal or contributory role of these viruses to the gross and histologic lesions of this sea lion remains to be determined.

Causes of swine polyserositis in a high-density breeding area in Italy.

We assessed the causes of polyserositis in pigs, categorized by causative agents and ages of animals affected. In a 3-y study, 246 pigs from 80 different farms with recurrent problems of polyserositis, in a high-density breeding area, were submitted for autopsy; 154 pigs with typical fibrinous serosal lesions were sampled for further bacterial and viral investigation. The most common gross lesions were pleuritis and pericarditis (141 of 154; 92%). The animals most affected were weaned pigs (139 of 154; 90%). Haemophilus parasuis and Mycoplasma hyorhinis were the most common bacteria detected and were present at the same rate (85 of 154; 55%). Other bacteria isolated were Streptococcus sp. (44 of 154; 29%), Pasteurella multocida (21 of 154; 14%), Escherichia coli (19 of 154; 12%), Actinobacillus pleuropneumoniae (7 of 154; 5%), and Trueperella pyogenes (4 of 154; 3%). Porcine reproductive and respiratory syndrome virus (PRRSV; 119 of 154; 77%) predominated among the viruses detected, followed, with lesser prevalence, by porcine circovirus 2 (40 of 154; 26%) and swine influenza A virus (19 of 154; 12%). Bacterial coinfection and coinfection of bacteria and viruses were common (128 of 154; 83%). A strong positive correlation was found between coinfection by H. parasuis and M. hyorhinis and also by H. parasuis with PRRSV.

Aspectos patológicos e imuno-histoquímicos da polisserosite em búfalos (Bubalus bubalis) / Histopathological and immunohistochemical study of the polyserositis in buffalo (Bubalus bubalis)

Polisserosites são alterações inflamatórias das serosas viscerais e parietais das cavidades corpóreas. Um tipo especial destas alterações foi identificado em bubalinos abatidos para consumo nos anos 80, sendo associada a infecção por Chlamydia psittaci. Apesar da importância da bubalinocultura no Pará, do caráter zoonótico da C. psittaci e da possibilidade de envolvimento de outros agentes na afecção, são raros trabalhos sobre a enfermidade desde os estudos pioneiros. No presente trabalho casos identificados como polisserosite pelo serviço de inspeção sanitária em búfalos abatidos para consumo foram coletados com objetivo de determinar a frequência e origem dos mesmos e de caracterizar as lesões e pesquisar antígenos de Chlamydia spp. nas mesmas. De um total de 2.887 bubalinos abatidos no período de estudo foram identificados 48 (1,66%) casos. O município de Santa Cruz do Arari na Ilha de Marajó apresentou a maior porcentagem de casos em relação ao número de animais abatidos (6,49%, 5/77). Na macroscopia as lesões se caracterizaram por áreas opacas, branco-amareladas de espessamento das serosas, por vezes com franjas fibrosas na superfície. Os resultados permitem concluir que os casos classificados como polisserosite pelos veterinários da inspeção sanitária nos búfalos abatidos para consumo correspondem na histopatologia a um tipo incomum de polisserosite, caracterizada por infiltrado linfocitário, com ocasionais formações de folículos linfoides terciários ou ectópicos. Foi observado ainda nestas lesões um predomínio de linfócitos T (CD3 positivos) no infiltrado, sendo os linfócitos B (CD79 positivos) presentes em maior número no interior das estruturas foliculares. Não foram demonstrados antígenos de clamídias nas lesões do presente estudo, o que demonstra a necessidade de estudos adicionais relativos a(s) etiologia(s) das lesões.(AU)

Polyserositis are inflammatory changes of the visceral and parietal serous of body cavities. A special type of polyserositis was identified in buffaloes in the 80s, being associated with infection by Chlamydia psittaci. Since these pioneering studies, there are no additional works about the condition. Considering the importance of buffalo in Pará, the zoonotic character of C. psittaci and the possibility of involvement of other agents in polyserositis in buffaloes the present study is proposed. We collected cases identified as polyserositis by sanitary inspection service in buffalo slaughtered for consumption in Belem for a complementary characterization of inflammatory cell and the research of Chlamydia spp antigens in lesions. Of 2.887 buffaloes slaughtered in a period of six months, there were 48 (1.66%) cases of polyserositis and 39 analyzed. Santa Cruz do Arari in Marajó Island was the city with the highest frequency of cases, whereas 6.49% of buffaloes had lesions. However, 50% of the present study cases came from Soure municipality in Marajó Island, which provided about 49% of buffaloes slaughtered in the period. In the macroscopy, there were opaque areas with white-yellow thickening of the serous, sometimes with fibrous fringes on the surface. Histopathology showed connective tissue projections partially lined by cuboid or flattened mesothelial cells. Often in projections there were mononuclear infiltrate of variable intensity, consisting mainly of lymphoid cells, with occasional ectopic or tertiary lymphoid follicles.(AU)

Efficacy of an inactivated Mycoplasma hyorhinis vaccine in pigs.

Lameness and polyserositis in pigs caused by Mycoplasma hyorhinis are generally treated with antibiotics and may require multiple doses. The costs of these antibiotics combined with economic losses from culling and reduced feed conversion due to lameness are hardships to the swine producer. In this study we have demonstrated efficacy of an inactivated M. hyorhinis vaccine administered to three-week old caesarian-derived colostrum-deprived piglets. Three doses of vaccine (high, medium, and low) were evaluated and compared to a placebo control. Mycoplasma hyorhinis challenge occurred three weeks after vaccination. Pigs were observed for lameness and respiratory distress for three weeks following challenge. Pigs were then euthanized and a gross pathological evaluation for polyserositis and arthritis was performed. A minimum immunizing dose of vaccine was defined as containing at least 7.41 × 107 CCU of M. hyorhinis per 2.0 mL dose as represented by the medium dose vaccine. This vaccine provided significant reductions in lameness and pericarditis with preventive fractions of 0.76 (95% CI [0.26, 0.92]) and 0.58 (95% CI [0.31, 0.74]), respectively, compared to the placebo control group. A significant increase in post-challenge weight gain (P < .0001) was also achieved with this vaccine, with an average daily gain (ADG) of 0.92 lbs/day compared to 0.57 lbs/day in the placebo group.

Antibacterial, anti-inflammatory, and antioxidant effects of Yinzhihuang injection.

The Yinzhihuang injection, a traditional Chinese medicine, has been the recent target of increasing interest due to its anti-inflammatory properties. The molecular basis by which Yinzhihuang injection could cure Riemerella anatipestifer (RA) serositis in ducks is unclear. This study evaluated the antibacterial, anti-inflammatory and antioxidant effects of Yinzhihuang injection, using disease models of RA-induced infectious serositis in ducks and heptane-induced inflammation in mice and rats. The duck mortality rate was reduced from 60% to 20% and both the inflammatory response and histological damage were ameliorated by treatment with Yinzhihuang injection (0.02 g/kg). Further studies indicated that superoxide dismutase (SOD), nitric oxide synthase (NOS), and inducible nitric oxide synthase (iNOS) were elevated while malondialdehyde (MDA), nitric oxide (NO) and RA growth were inhibited when the ducks were treated by Yinzhihuang injection. In addition, Yinzhihuang injection (0.04 g/ml) effectively inhibited xylene-induced auricle swelling in mice, (demonstrating an inhibition rate of 35.21%), egg albumen-induced paw metatarsus swelling in rats, (demonstrating an inhibition rate of 22.30%), and agar-induced formation of granulation tissue. These results suggest that Yinzhihuang injection ameliorates RA-induced infectious serositis in ducks by modulation of inflammatory mediators and antioxidation.

Haemophilus parasuis serovars isolated from pathological samples in Northern Italy.

From January 2007 to December 2011, a total of 106 Haemophilus parasuis strains isolated from pigs were serotyped by agar gel diffusion test (DG). Serovar 4 was the most prevalent (24.5%), followed by serovar 13 (19.8%) and serovar 5 (11.3%). Twenty-nine strains were non-typeable (27.3%). The strains were divided into two groups, depending on whether they were isolated from specific pathological lesions of systemic disease such as polyserositis, arthritis or meningitis (73 cases of 106) or from the lower respiratory tract of pigs suffering from bronchopneumonia (33 cases of 106). Serovars 4 and 13 had a higher prevalence in systemic infection (polyserositis) than in respiratory disease only. Pasteurella multocida (14/106), Streptococcus suis (7/106), Actinobacillus pleuropneumoniae (4/106), Bordetella bronchiseptica (3/106) and Arcanobacterium pyogenes (3/106) were isolated in association with H. parasuis.

Complete genome sequence of Riemerella anatipestifer reference strain.

Riemerella anatipestifer is an infectious pathogen causing serositis in ducks. We had the genome of the R. anatipestifer reference strain ATCC 11845 sequenced. The completed draft genome consists of one circular chromosome with 2,164,087 bp. There are 2,101 genes in the draft, and its GC content is 35.01%.

Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis.

An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.

Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis.

The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs.

Distribution of capsular serotypes and virulence markers of Streptococcus suis isolated from pigs with polyserositis in Korea.

The objective of this study was to determine the capsular serotypes and potential virulence factors of Streptococcus suis isolated from pigs with polyserositis. Among the 24 isolates evaluated, serotype 3 [7 (29%) of the isolates] and serotype 4 [5 (21%)] were the most common. The isolates were also studied for the presence of the genes mrp, epf, and sly, which encode muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY), respectively. Of the 24 isolates, 8 carried mrp: 4 of serotype 3, 2 of serotype 2, and 2 of serotype 4. One mrp(+) isolate (serotype 2) also carried the epf gene. All 24 isolates carried the sly gene. The serotype and genotype distribution greatly differed from that reported for isolates from pigs with other clinical manifestations of S. suis infection in other countries.

Protection of vaccinated pigs against experimental infections with homologous and heterologous Haemophilus parasuis.

The efficacy of a new Haemophilus parasuis vaccine for pigs was investigated. The vaccine contains H parasuis serotype 5 cells and is adjuvanted with Diluvac Forte (Intervet). Groups of pigs were vaccinated at five and seven weeks with 2 ml and their littermates served as unvaccinated controls. The vaccinated pigs were protected against a challenge with another strain of Hparasuis serotype 5 at two, eight and 17 weeks after the second vaccination, whereas the controls became very ill. The susceptibility of the pigs to the infection decreased with increasing age. After a heterologous challenge with H parasuis serotypes 1, 12, 13 and 14, two weeks after the second vaccination, the vaccine also gave clear protection. The severity of the illness among the control pigs differed with the different serotypes.

Effect of vaccinating sows and their piglets on the development of Glässer's disease induced by a virulent strain of Haemophilus parasuis serovar 5.

Ten pregnant gilts were divided into two groups of five and one group was vaccinated at 80 and 95 days of pregnancy with a commercial bacterin containing Haemophilus parasuis serovars 2, 3 and 5. Half the piglets born to each group of gilts were vaccinated at seven and 21 days of age with the same bacterin, and one week after they were weaned at five weeks, all the piglets were inoculated intratracheally with 10(6) colony-forming units of Hparasuis serovar 5. At slaughter, a significantly smaller percentage of the lungs of the pigs born to the vaccinated gilts was affected by pneumonic lesions, and significantly fewer of them had arthritic joint changes. The average daily liveweight gain of the pigs born to the vaccinated gilts was significantly greater than that of those born to the unvaccinated gilts, but the vaccination of the piglets had no effect. There was no significant difference between the feed conversion ratios of the four groups of piglets, and none between the average times they took to reach slaughter weight. The pigs born to the vaccinated gilts had higher ELISA titres to Hparasuis than those born to the unvaccinated gilts.

The effect of a homologous bacterin given to sows prefarrowing on the development of Glässer's disease in postweaning pigs after i.v. challenge with Haemophilus parasuis serotype 5.

UNLABELLED: The trial was carried out to evaluate the impact of maternal antibodies on the development of Glässer's disease after i.v. exposure of weaned pigs with a homologous serovar of Haemophilus parasuis (HPS). Two groups of weaned pigs were formed. Group one VI (n = 10): born to vaccinated sows, weaners i.v. challenged one week postweaning and euthanatized 14 days postweaning. Group two NVI (n = 10 wearners): born to non vaccinated sows, i.v. challenged one week postweaning euthanatized 14 days postweaning. One week postweaning all weaners were i.v. inoculated with HPS serovar 5. The following parameters were evaluated: clinical signs (depression, centralnervous signs, fever, lameness), macroscopic lung, pleura, peritoneum, liver and joint changes, and mortality. All trial sows were HPS seronegative prior to vaccination. The HPS vaccinated sows were proven seropositive on day 3 p.p. (values > 0.24), the non vaccinated ones were tested seronegative (values < 0.23). The progeny of sows vaccinated prefarrowing with two doses of HPS serovar 5 bacterin were partially protected against HPS caused clinical and pathological signs. The majority of clinical signs as fever, depression, recumbency, lack of response to verbal stimuli and lameness showed significant (P < 0.05) milder clinical symptoms in VI than in NVI animals. Respiratory signs (P = .169) and involvement of the central nervous system as ataxia, muscular tremor, incoordination of hind legs and convulsions (P = 1) showed no significant differences between the groups. Except lesions of pericard (VI vs. NVI, P = .14) and pleura (VI vs. NVI, P = .14) there were significant (P < 0.05) macroscopic differences at necropsy in lung, liver, joints and cerebrospinal fluid between the offspring of vaccinated sows and the ones of non vaccinated dams. No HPS were isolated from the nasal mucosa of the pigs prior to inoculation. HPS serovar 5 was recovered at necropsy from the nasal mucosa of all pigs in both groups. One pig from group VI presented in all examined organs the presence of HPS serovar 5. The remaining animals in group VI revealed in lung, pericard, pleura, liver, joints and cerebrospinal fluid no presence of HPS. The rate of isolation between VI and NVI groups revealed a significant (P < 0.05) difference. All the survived piglets of group NVI showed positive ELISA titres against HPS serovar 5 (values > .24). The piglets that died or were euthanatized before the end of the study have not been subjected to ELISA serological testing. One piglet died in group VI before the end of the study. Non of the remaining animals in group VI showed seroconversion to HPS serovar 5. IMPLICATIONS: Vaccination of sows did not influence the colonisation of nasal mucosa, but progeny of sows vaccinated prefarrowing with two doses of HPS serovar 5 bacterin were partially protected against HPS caused diseases.

Thelebolus microsporus mycelial mats in the trachea of wild brown skua (Catharacta antarctica lonnbergi) and South Polar skua (C. maccormicki) carcasses.

Sixteen brown skuas (Catharacta antarctica lonnbergi) and seven South Polar skuas (C. maccormicki) were found dead near Boekella Lake, Hope Bay, Antarctica, in February 1997. Postmortem examination revealed conspicuous caseous, deep yellow fungal/mycelial mats or cores in the trachea of nine of 19 carcasses that were examined. These mycelial cores, highly suggestive of aspergillomas, completely occluded the tracheal lumen in four of these nine carcasses. Thelebolus microsporus, a psychrophilic ascomycetous fungus commonly isolated from skua dung and skua nesting material, was isolated in pure culture from these tracheal plugs. Awareness of pseudolesions resulting from Thelebolus microsporus profuse postmortem growth in the trachea of dead skuas will minimize potential confusion with aspergillosis when investigating causes of epornithics in Antarctica.

Mycoplasma alligatoris sp. nov., from American alligators.

Mycoplasmas were isolated from multiple tissues of diseased American alligators (Alligator mississippiensis). This paper presents biochemical, serological and molecular genetic characterizations of a lethal pathogen of alligators for which the name Mycoplasma alligatoris sp. nov. is proposed. The type strain is A21JP2T (ATCC 700619T).

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques (Macaca mulatta).

CONTEXT: Enterocytozoon bieneusi is the most frequent microsporidian parasite of human patients with acquired immunodeficiency syndrome and is a significant cause of diarrhea and wasting. Recently, this organism has also been recognized as a spontaneous infection of several species of captive macaques. As in humans, E bieneusi frequently causes enteropathy and cholangiohepatitis in immunodeficient simian immunodeficiency virus (SIV)-infected macaques. OBJECTIVE: To examine E bieneusi as an etiologic agent of nonsuppurative proliferative serositis in immunodeficient rhesus macaques (Macaca mulatta). DESIGN: Retrospective analysis of necropsy material obtained from immunodeficient SIV-infected rhesus macaques. RESULTS: Examination of SIV-infected rhesus macaques (n = 225) revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin. The organism could be identified by in situ hybridization and polymerase chain reaction in sections of pleura and peritoneum obtained at necropsy. Serositis was always accompanied by moderate-to-severe infection of the alimentary tract, and morphologic evidence suggested dissemination through efferent lymphatics. Colabeling experiments revealed most infected cells to be cytokeratin positive and less frequently positive for the macrophage marker CD68. Sequencing of a 607-base pair segment of the small subunit ribosomal gene revealed 100% identity to sequences obtained from rhesus macaques (Genbank accession AF023245) and human patients (Genbank accession AF024657 and L16868). CONCLUSIONS: These findings indicate that E bieneusi disseminates in immunodeficient macaques and may be a cause of peritonitis in the immunocompromised host.