Colonization of rabbits with Staphylococcus aureus in flocks with and without chronic staphylococcosis.

Rabbits of 19 rabbitries were examined for the presence of Staphylococcus aureus in nine different body sites. Seven rabbitries experienced epidemically spreading signs of staphylococcosis while the other 12 rabbitries did not. S. aureus was isolated in all seven flocks that suffered from chronic problems of staphylococcosis and in 11 of the 12 clinically healthy flocks. The mean percentage of infected animals in these two groups was 90 and 43.3%, respectively. S. aureus was isolated from all body sites examined, but the ear and the perineum were often more intensely colonized. The number of animals colonized with S. aureus and the mean number of positive body sites in S. aureus positive rabbits were significantly higher in rabbitries with chronic staphylococcosis. This indicates that colonization capacity of S. aureus plays a role in epidemically spreading disease in rabbits. S. aureus isolates belonged to five different biotypes and 23 different phage types. Several different types simultaneously circulated in contaminated rabbitries and even simultaneously infected individual rabbits. Strains that belonged to the biotype-phage type combination mixed CV-C, 3A/3C/55/71 only occurred in rabbitries chronically dealing with signs of staphylococcosis. This may indicate a relationship between phenotypic strain properties and virulence of S. aureus.     

High and low virulence Staphylococcus aureus strains in a rabbit skin infection model

In the present study, an in vivo rabbit skin infection model was developed to reproduce the lesions caused by high and low virulence Staphylococcus aureus strains from rabbits. "O"-shaped dermal skin lesions were induced on the shaved flanks of anaesthetised rabbits using a tattoo pin and pincers. The induced lesions on the flanks of four groups of 10 rabbits were then inoculated by topical application of 0.1 ml of 10(8)cfu S. aureus bacteria. One group was inoculated with a typical high virulence (HV) S. aureus strain from rabbits, one group received an atypical HV strain and two groups were inoculated with low virulence (LV) strains. Five animals were kept as negative controls. The development, appearance and size of abscesses were scored daily for a period of 2 weeks. The infection model showed reproducible results for the different S. aureus inoculation groups. Inoculation of the skin with the typical HV strain resulted in significantly larger abscesses than those caused by the LV strains. The atypical HV strain caused abscesses of a size intermediate to that obtained with the HV and LV strains. In rabbits infected with LV strains, most of the lesions had healed by day 14 post-inoculation. The devised infection model is able to reliably reproduce the virulence properties of HV and LV S. aureus strains.     

Differentiation between high and low virulence Staphylococcus aureus strains from rabbits by randomly amplified polymorphic DNA (RAPD) analysis

Randomly Amplified Polymorphic DNA (RAPD) typing was performed on 53 rabbit Staphylococcus aureus strains. Twenty-three strains isolated in 13 different rabbitries with chronic problems of staphylococcosis, showed the same RAPD banding pattern. Twenty of these strains belonged to the 'mixed CV-C' biotype and to the phage-type 3A/3C/55/71, previously described to be highly virulent in rabbits, and three strains belonged to other biotypes or phage-types. None of the strains isolated from rabbitries without chronic problems of staphylococcosis showed this specific RAPD pattern. RAPD analysis can be used as a rapid and reliable test method to differentiate between the characteristic genotype corresponding to high virulence and other S. aureus strains from rabbits. This is useful for the diagnosis and prevention of the introduction of these highly virulent strains in industrial rabbitries.     

Experimental infection of specific pathogen free piglets with French strains of Streptococcus suis capsular type 2.

A standardized model of Streptococcus suis type 2 infection in specific-pathogen-free piglets, housed in high-security barns, was used to compare the virulence of 3 French field strains of S. suis serotype 2 isolated from tonsils of a healthy pig (strain 65) or from diseased pigs (meningitis, strain 166', or septicemia, strain 24). In one of the 2 trials, 7-week-old pigs, in 3 groups of 8, were inoculated intravenously with 2 x 10(8) colony-forming units of S. suis type 2. In each group, 1 uninfected animal was a sentinel. Eight animals were also used as negative control group. The experiment was repeated under similar conditions with strains 65 and 166'. Virulence differed markedly among these S. suis strains when clinical signs, zootechnical performances, lesions, and bacteriological data were analyzed. Strain 65 did not induce clinical signs in inoculated pigs. In contrast, pigs infected with the other 2 strains exhibited clinical signs and typical lesions of S. suis type 2 infections. Differences in virulence were also observed between the 2 virulent strains. Sentinel animals exhibited the same manifestations as those recorded in inoculated piglets. Results were similar in the second trial, indicating that under the present experimental conditions, results were reproducible. The standardized conditions described in this study could be a useful tool to further study about the S. suis infection.     

Pasteurella multocida infection in the domestic rabbit: immunization with a streptomycin-dependent mutant.

Fourteen Pasteurella multocida-free rabbits were inoculated intranasally with a streptomycin-dependent mutant of P. multocida serotype 12:A. Vaccinations with approximately 10(8) colony forming units were done on days 0, 14 and 28. Two weeks later the animals were separated into groups, which included 12 rabbits divided into two control groups of six unvaccinated Pasteurella-free animals. Seven vaccinated rabbits were challenged intranasally with the homologous virulent parent strain and the other seven vaccinates were challenged with a virulent strain of serotype 3:A. Rabbits were necropsied two weeks later. The vaccinated group challenged with the parent strain showed a more rapid nasal clearance of the organism than the vaccinated group challenged with the heterologous strain. However, the number of positive cultures of P. multocida recovered from tissues post-challenge were similar in vaccinated and control animals. In a significant number of animals, vaccination with serotype 12:A induced detectable antibody production to somatic antigens of both 12:A and heterologous strain 3:A.     

Diarrhea associated with enterotoxigenic Bacteroides fragilis in foals

Enterotoxigenic Bacteroides fragilis (ETBF) was isolated from the feces of 10 of 40 Thoroughbred foals with naturally acquired diarrhea. Of the 10 foals positive for ETBF, 6 were less than or equal to 7 days old. Fecal specimens from 4 of the 10 foals also were positive for rotavirus, and one fecal specimen was positive for Salmonella enteritidis. Clinical or hematologic differences were not evident between foals infected with ETBF only and those infected with ETBF and another recognized enteric pathogen. Only 1 of 10 foals infected with ETBF died. Of 25 adult rabbits with ligated ceca, 23 developed mucoid, often hemorrhagic, diarrhea after inoculation of 5 X 10(9) viable ETBF cells into the ileum. Nine of 13 (69%) rabbits inoculated with 1 of 3 isolates of ETBF died, but none of 12 inoculated with 1 of 6 other isolates of ETBF died. Enteric disease did not develop in 15 rabbits inoculated with nonenterotoxigenic B fragilis.     

12株中国伊氏锥虫对小鼠的毒力试验

为了确定中国伊氏锥虫各株的毒力强弱,对中国伊氏锥虫:安徽水牛株(AHB)、广东阳江水牛株(GDB_1)、广东水牛株(GDB_2)、广东马株(GDH)、广西骡株(GXM)、湖北骡株(HBM)、湖南水牛株(HNB)、江苏高邮水牛株(JSB_1)、江苏盱眙水牛株(JSB_2)、新疆骆驼株(XJCA)、云南水牛株(YNB)、浙江水牛株(ZJB)进行小鼠的毒力试验。以各组鼠死亡率和平均存活天数作为毒力强弱的主要依据。结果表明最强致死率为100%,最弱30%;致死所需时间平均为7.5 d～25 d。对小鼠的致病力强弱依次是:AHB>YNB>GDB_2>XJCA>HBM>HNC>JSB_1>GXM>GDB_1>GDH>JSB_2>ZJB。提示不同株的毒力差异显著,测定结果可为伊氏锥虫相关科学研究选株提供依据。     

Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs.

Experimental airborne transmission of Streptococcus suis type 2 was studied in specific pathogen free piglets. Forty piglets were allotted to five groups of eight 7-week-old animals and housed in three separated units. Negative control pigs (group 1) were housed in unit A and infected batches were housed in units B (group 2) and C (groups 4). In units B and C, non-inoculated groups (groups 3 and 5, respectively), 40 cm distant from the respective inoculated group and without any physical contact between them, also took place. Six animals of groups 2 and 4 were inoculated intravenously with 2 x 10(8) colony forming units (cfu) of a mild and a high virulent S. suis strains, respectively. The remaining animals in these groups and pigs from groups 1, 3, 5 received broth medium in the same way. Differences among virulence of S. suis capsular type 2 were observed in inoculated pigs of groups 2 and 4. Pigs from group 2 became carriers, showing only mild symptoms. By contrast, animals from group 4 presented an acute form of the disease. All the indirect contact pigs in groups 3 and 5 had S. suis in palatine tonsils from day 6 after the infection and they presented clinical manifestations similar to those observed in experimentally infected pigs. Two direct contact animals were also contaminated in the upper respiratory tract but surprisingly they did not show any symptoms. Airborne transmission of S. suis in experimentally pigs was demonstrated in the present study. Indirect infections, as described in this study, are a more realistic way to infect pigs than other experimental procedures and may be used to further study the pathogenesis of the infection caused by this important pathogen.     

Detection and antigenicity of chlamydial proteins that bind eukaryotic cell membrane proteins.

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (CMP) of noninfected cells were labeled with biotin (B-CMP), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (EB) were reacted with the B-CMP extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound B-CMP. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound B-CMP. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 EB and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of a single milbemycin oxime administration in combination with praziquantel against experimentally induced heartworm (Dirofilaria immitis) infection in cats

The efficacy of a combination of milbemycin oxime and praziquantel in preventing the establishment of experimentally induced heartworm (Dirofilaria immitis) infection was investigated in a study involving 24 young domestic short-hair cats. The animals were inoculated with 50 infective larvae on day 0. Subsequently they were divided into two groups of 12 animals each. The animals in group 1 were treated once with medicated tablets containing 4 mg milbemycin (minimum dose 2 mg/kg body weight) and 10 mg praziquantel (MILBEMAX) on day 30 after infection. Cats in group 2 received placebo tablets on the same day. On day 183 post-infection a blood sample was taken from each animal before euthanasia and necropsy. The blood samples were tested for the presence of microfilariae and the necropsied animals were examined for the presence of adult worms. Microfilariae were not found in any of the investigated cats. No heartworms were found in the animals in group 1 (treated with medicated tablets). Out of the 12 placebo-treated cats 1 was heartworm-free, whereas all the others were found to be infected with 1-3 adult heartworms.     

Experimental mastitis caused by Mycoplasma bovigenitalium and M. canadense in the ewe

Twelve strains of M. bovigenitalium and two of three strains of M. canadense caused an infection resulting in a pathogenic effect when experimentally inoculated into the ovine mammary gland. Differences in the pathogenesis were quantified by the duration of continuous mycoplasma excretion and the duration of high milk cell levels, but variation in the susceptability of the experimental animals prevented the establishment of firm conclusions on the relative virulence of the strains. Seven M. bovigenitalium and two M. canadense strains were eventually eliminated naturally from the infected glands, but four M. bovigenitalium strain infections ultimately became sub-clinical with intermittent mycoplasma excretion and low milk cell levels.     

Transmission of ovine herpesvirus 2 among adult sheep

Previous studies from this laboratory have defined the pattern of acquisition of ovine herpesvirus 2 (OHV-2) in lambs under natural flock conditions. This study examined the question of whether OHV-2 could be transmitted between adult sheep. Two potential routes of transmission were examined: (1) direct inoculation of either viable leukocytes or whole blood from OHV-2 positive sheep, and (2) horizontal transmission through natural contact with OHV-2 positive sheep. Two groups of OHV-2 negative adult sheep were inoculated with material from infected sheep, one with 5x10(8) viable peripheral blood leukocytes (PBL), and the other with 100 ml of whole peripheral blood. No PCR signals were detected in any of the three sheep inoculated with the PBL during the 20 weeks following inoculation. In the group of five sheep inoculated with whole blood, two became PCR-positive at 7 and 8 weeks post-inoculation, respectively, and the remaining three sheep maintained their negative status until termination of the experiment at 20 weeks post-inoculation. In two experiments conducted in different flocks, a total of 20 adult sheep were used to examine horizontal transmission by contact; all animals became PCR-positive within 12 months of mixing the uninfected and infected animals. The results of these experiments support two conclusions. First, the susceptibility to OHV-2 is not limited to young lambs; adult sheep remain fully susceptible. Second, the fact that whole blood, but not PBL, from infected sheep was able to transmit the infection to only two of five inoculated sheep suggests that the infection in peripheral blood cells may be largely non-productive.     

Failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus

Previous reports on the spread of bovine virus diarrhoea virus (BVDV) from animals primarily infected with the agent are contradictory. In this study, the possibility of transmission of BVDV from calves simultaneously subjected to acute BVDV and bovine coronavirus (BCV) infection was investigated. Ten calves were inoculated intranasally with BVDV Type 1. Each of the 10 calves was then randomly allocated to one of two groups. In each group there were four additional calves, resulting in five infected and four susceptible calves per group. Virulent BCV was actively introduced in one of the groups by means of a transmitter calf. Two calves, susceptible to both BVDV and BCV, were kept in a separate group, as controls. All ten calves actively inoculated with BVDV became infected as shown by seroconversions, and six of them also shed the virus in nasal secretions. However, none of the other eight calves in the two groups (four in each) seroconverted to this agent. In contrast, it proved impossible to prevent the spread of BCV infection between the experimental groups and consequently all 20 study calves became infected with the virus. Following infection, BCV was detected in nasal secretions and in faeces of the calves and, after three weeks in the study, all had seroconverted to this virus. All calves, including the controls, showed at least one of the following clinical signs during days 3-15 after the trial started: fever (> or =40 degrees C), depressed general condition, diarrhoea, and cough. The study showed that BVDV primarily infected cattle, even when co-infected with an enteric and respiratory pathogen, are inefficient transmitters of BVDV. This finding supports the principle of the Scandinavian BVDV control programmes that elimination of BVDV infection from cattle populations can be achieved by identifying and removing persistently infected (PI) animals, i.e. that long-term circulation of the virus without the presence of PI animals is highly unlikely.     

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics,clinical outcome and peripheral and local immune responses

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 10⁷ tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.     

Experimental infection of laboratory animals and sheep with Gongylonema pulchrum in Japan

Japanese White rabbits, Wistar rats, ddY mice, Suffolk sheep, and a domestic cat were each orally inoculated with 20-140 third-stage larvae (L3) of Gongylonema pulchrum, isolated from naturally infected dung beetles captured in Aomori Prefecture. Worm recovery rates were 40.0-72.0% in rabbits at 7, 14, and 19 weeks post-infection (PI) and 3.3-25.0% in rats at 19 weeks PI. Those in 2 sheep at 7 weeks PI showed 53.6% and 29.3%. No worms were recovered from the mice and the cat. In the susceptible animals, many worms were found in the esophagus, and a few were present in the pharyngeal mucosa, tongue, buccal mucosa, and cardiac portion of the stomach wall. No distinct morphological differences were observed in the worms from rabbits and sheep. These results indicate that rabbits are very suitable experimental definitive hosts for G. pulchrum.     

Ultrastructural pathology of nasal and tracheal mucosa of rabbits experimentally infected with Pasteurella multocida serotype D:1

Sixteen 8- to 9-week-old Pasteurella multocida-free New Zealand White rabbits were divided into two equal groups. The first group was inoculated intranasally with P multocida serotype D:1 strain and the second group that was inoculated with phosphate-buffered saline (PBS) only was used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and from tracheal swabs of seven rabbits in this group. Four rabbits in group 1 died with clinical signs of septicaemia, two rabbits had mucopurulent nasal discharge and pneumonic lesions and the other two did not show any clinical signs or gross lesions. The ultrastructural changes detected were deciliation or clumping of cilia of ciliated epithelium, cellular swelling, vacuolation and sloughing. The subepithelial capillaries showed congestion, intravascular fibrin deposition, platelets aggregation and endothelial injury. Pasteurella multocida was observed attached to the injured endothelial cells. Heterophils, mast cells, vacuolated monocytes and macrophages infiltrated the lamina propria and between the degenerated epithelial cells.     

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.     

Antimicrobial resistance and resistance genes in Staphylococcus aureus strains from rabbits

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.     

Prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens among high and low virulence rabbit Staphylococcus aureus strains

Staphylococcus aureus is an important cause of pododermatitis, subcutaneous abscesses and mastitis in rabbits. On rabbitry level, two types of S. aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection affects only a small number of animals. The second type of infection is caused by high virulence strains and spreads throughout the rabbit flock. The pathogenic capacity of a particular S. aureus strain is attributed to a combination of invasive properties and extracellular factors such as toxin production. Therefore, 22 high virulence and 37 low virulence S. aureus isolates were compared regarding the prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens. This study revealed a clearly significant difference between HV and LV rabbit S. aureus strains. All typical HV isolates were positive for the egc cluster, containing the enterotoxin(like) genes seg, sei, selm, seln, selo and selu, whereas these genes were not detected in any of the LV isolates. Further research is necessary to clarify the importance of the egc cluster in the pathogenesis of infections with high virulence S. aureus strains in rabbits.     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.