Protection of macaques against intrarectal infection by a combination immunization regimen with recombinant simian immunodeficiency virus SIVmne gp160 vaccines

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.     

Limited Breadth of the Protective Immunity Elicited by Simian Immunodeficiency Virus SIVmne gp160 Vaccines in a Combination Immunization Regimen

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.     

Immunogenicity and protective efficacy of Gag/Pol/Env vaccines derived from temporal isolates of SIVmne against cognate virus challenge

BACKGROUND: We used the SIVmne model to examine the relative immunogenicity and protective efficacy of vaccines derived from temporal isolates of lentivirus infection. SIVmne170 is a molecular clone isolated from a pig-tailed macaque 170 weeks after inoculation with SIVmneCL8. METHODS: We immunized pig-tailed macaques with Gag/Pol/Env vaccines derived from CL8 or 170 and examined their protective efficacy against CL8, 170, or chimeras 8/170 and 170/8, containing the 5' or 3' half of the respective parental genomes. RESULTS: As expected, CL8 vaccines protected animals against the CL8, but not the 170 virus. Surprisingly, 170 vaccines not only failed to protect against the 170 virus, but also the less pathogenic CL8. Chimeric virus challenges revealed that the envelope antigen of CL8 represents an important target for protective immunity. CONCLUSIONS: These results underscore the potential importance of targeting transmitted viruses through judicious choice of immunogens from early isolates for vaccine development.     

Role of Immune Responses against the Envelope and the Core Antigens of Simian Immunodeficiency Virus SIVmne in Protection against Homologous Cloned and Uncloned Virus Challenge in Macaques

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.     

Evaluation of protective efficacy of recombinant subunit vaccines against simian immunodeficiency virus infection of macaques.

Simian immunodeficiency virus (SIV) was used as a model to study the protective efficacy of an immunization regimen currently being evaluated as candidate vaccines against HIV in human subjects. Four Macaca fascicularis were first immunized with recombinant vaccinia virus expressing the envelope glycoprotein gp160 of SIVmne and then boosted with subunit gp160. Both cell-mediated and humoral immune responses against SIV, including neutralizing antibodies, were elicited. The macaques were shown to be protected from a homologous virus infection as determined by serology, lymphocyte cocultivation, polymerase chain reactions and in vivo transmission analyses. Four unimmunized control animals were readily infected. However, viremia in infected control animals could decrease substantially following the initial phase of infection so that persistent infection might not be readily detectable.     

Capture and transfer of simian immunodeficiency virus by macaque dendritic cells is enhanced by DC-SIGN

Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4(+), coreceptor-positive cells in trans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4(+), coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4(+) T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.     

Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.     

Trypanosoma congolense: Natural and acquired resistance in the bovine

A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.     

Mode of infection of Hokkaido virus (Genus Hantavirus) among grey red-backed voles, Myodes rufocanus, in Hokkaido, Japan

Hokkaido virus (HOKV) is a member of the genus Hantavirus, in the family Bunyaviridae. To investigate HOKV infection in the host Myodes rufocanus, the grey red-backed vole, 199 animals were captured at Tobetsu (October 2004 and July 2005) and Nakagawa (October 2004) in Hokkaido, Japan, for detection of antibody, antigen, and viral RNA. In the surveys in Tobetsu (2004) and Nakagawa (2004), seropositive animals were detected at a frequency of 6.0% (5/84) and 10.4% (5/48), respectively. No seropositive animals were detected in Tobetsu in 2005. Seroprevalence in males in Tobetsu and Nakagawa in 2004 was 25% (1/4) and 45.5% (5/11), respectively, which was higher than in females, at 5.0% (4/80) and 0% (0/37), respectively (P<0.01). These results suggest that male animals play an important role in the maintenance of HOKV in M. rufocanus. Two females were seronegative but viral RNA-positive, indicating that these animals had acute infections before antibody was produced. Another five infected animals in Nakagawa were all male and had high levels of antibodies and viral RNA, suggesting that they had persistent infections. Viral RNA copies in organs of infected animals in Nakagawa were quantified by real-time polymerase chain reaction. Two acutely infected animals had > or = 10 times the number of RNA copies in their lungs compared to those of persistently infected animals. In most cases, lungs or spleen had the highest RNA copy number, regardless of infection status.     

Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor.

Myxoma virus (MYX) is a leporipoxvirus of rabbits that induces a lethal syndrome characterized by disseminated tumorlike lesions, generalized immunosuppression, and secondary gram-negative bacterial infection. A MYX deletion mutant (vMYX-GF- delta M11L) was constructed to remove the entire myxoma growth factor (MGF) coding sequence and that for the C-terminal five amino acids of the partially overlapping upstream gene, M11L. Unexpectedly, this deletion completely abrogates the capacity of MYX to cause the characteristic disease symptoms of myxomatosis. Upon inoculation of rabbits with vMYX-GF- delta M11L, recipient animals developed only a benign, localized nodule reminiscent of a Shope fibroma virus-induced tumor in which a single primary lesion appeared at the site of injection and then completely regressed within 14 days, leaving the animals resistant to challenge with wild-type MYX. No evidence of the purulent conjunctivitis and rhinitis that always accompany wild-type MYX infection was observed. To ascertain whether the attenuation observed in vMYX-GF- delta M11L was due to a combined effect of the MGF deletion and alteration of the upstream M11L gene, two additional MYX recombinants were constructed: an MGF- virus (vMYX-GF-) containing an intact M11L gene and an M11L- virus (vMYX-M11L-) containing an intact MGF gene. Infection with vMYX-GF- resulted in moderated symptoms of myxomatosis, but all clinical stages of the disease were still detectable. In contrast, disruption of M11L alone dramatically reduced the virus virulence, resulting in a nonlethal syndrome whose clinical course was nevertheless distinct from that of vMYX-GF- delta M11L. Upon inoculation with vMYX-M11L-, rabbits developed primary and secondary tumors which were larger and more circumscribed than those of wild-type MYX recipients. Whereas wild-type MYX infection always includes severe, purulent conjunctivitis and rhinitis, vMYX-M11L- recipients remained healthy and displayed only minimal signs of respiratory distress. By about 30 days after infection, the tumors induced by vMYX-M11L- had completely regressed and these animals were immune to challenge with wild-type MYX. Histological analysis indicated that tumors induced by vMYX-M11L- are much more heavily infiltrated with macrophages and heterophils and that the sites of viral replication are more edematous and necrotic than those of wild-type infection, suggesting that the host was able to mount a more vigorous inflammatory response to vMYX-M11L- infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.     

Infections caused by Mycobacterium avium subspecies avium, hominissuis, and paratuberculosis in free-ranging red deer (Cervus elaphus hippelaphus) in Austria, 2001-2004

Between 2001 and 2004, 14 Austrian free-ranging red deer (Cervus elaphus hippelaphus) infected by Mycobacterium avium species were observed. Eight of the cases were from different geographical regions, and six originated from the same hunting area. The affected animals had signs of diarrhea, severe weight loss, and emaciation. On post-mortem examination, lymphadenitis associated with grossly enlarged mesenteric lymph nodes as well as multiple caseous or purulent nodular lesions in the thickened wall of the intestines were present in all animals. In 10 cases M. avium subsp. avium and in four cases M. a. hominissuis were isolated. In three red deer, a mixed infection with M. a. hominissuis and M. a. paratuberculosis was evident. Typing of M. a. avium and M. a. hominissuis isolates was performed by polymerase chain reaction (PCR) detection of insertion sequence IS901 and the virulence-associated macrophage-induced gene (mig), inverted repeat (IR) typing (IS1245/IS1311), and random amplified polymorph DNA (RAPD) analysis. While all M. a. avium and M. a. hominissuis contained the mig gene, IS901 was detected only in M. a. avium. The prevalence of IS901-positive isolates correlated well with the geographic location of affected animals. The IS901-containing isolates were shown to be genotypically closely related, as they exhibit similar patterns in IR-typing and in RAPD analysis. In contrast, IS901-negative isolates (M. a. hominissuis) displayed distinct profiles in both molecular systems.     

Immunization against SIVmne in macaques using multigenic DNA vaccines

All structural and regulatory genes of SIVmne were cloned into mammalian expression vectors to optimize expression in vitro and immunogenicity in mice. Macaca fascicularis were immunized four times with plasmid DNA (n = 4), or two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag-Pol particles (n = 4). Following intrarectal challenge with SIVmne, all macaques became infected. Three monkeys immunized with DNA alone maintained low plasma virus loads by 1 year post-challenge; the fourth exhibited high virus loads and significant CD4+ cell decline. Two of the DNA plus boost and three control macaques had high virus loads and associated CD4+ cell decline. Both vaccine protocols elicited antibodies and comparable helper T-cell proliferative responses to gp160. Cytokine mRNA levels in activated peripheral blood mononuclear cells (PBMC) taken at time of challenge suggested a dominant T helper (Th) 1 state in three DNA-immunized and one protein-boosted macaque, which correlated with low virus loads and high CD4+ cell counts post-challenge.     

SIV of stump-tailed macaque (SIVstm) is a divergent Asian isolate.

Analysis of molecularly cloned DNAs of SIVs isolated from Asian rhesus macaque (Macaca mulatta; SIVmac) and pig-tailed macaque (Macaca nemestrina; SIVmne) has indicated a high degree of sequence homology between these viruses. Thus SIVmac and SIVmne might have originated from the same or very closely related viruses. We have cloned and sequenced a PCR-amplified segment containing the LTR sequences of SIV originating from a stump-tailed macaque (Macaca arctoides; SIVstm). Comparative sequence analysis indicates that SIVstm belongs to the SIV/HIV-2 group; however, it is genetically distinct from the other members.     

Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.     

Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters

Sweetfish, Plecoglossus altivelis, obtained from 18 rivers in Shizuoka Prefecture were examined for metacercarial infection of 2 flukes, Metagonimus yokogawai and Metagonimus miyatai. The infection rate and density of metacercariae in the fish were higher in eastern and western regions than in central region of the prefecture. After infection of hamsters with metacercariae derived from the scale, 98.7% of the adult worms obtained from the intestine was found to be M. miyatai. Conversely, from infection with metacercariae from the flesh, 90.0% of the worms was M. yokogawai. Since the worms had no exclusivity in the tissues, we conclude that the flukes have location preference with the former primarily preferring the scale, and the latter the flesh. Fish from two rivers located in adjacent areas in the western region had relatively a higher ratio of M. yokogawai in the scale relative to other rivers, suggesting an intraspecific genetic variation due to geographical isolation. On examination of adult worms in the hamster's intestine, M. yokogawai was mainly located towards the anterior part of the intestine, unlike M. miyatai, suggesting that in mammalian host too, the parasites have site preference.     

Changes in hydrolytic enzyme activities in armadillos infected with Mycobacterium leprae

The activities of hydrolytic enzymes in various organs of armadillos infected with Mycobacterium leprae were compared with those in normal armadillos. Except for aspartate aminopeptidase and esterase, the levels of the other enzymes in liver, spleen and inguinal lymph nodes were significantly higher in armadillos infected with M. leprae compared with those in non-infected ones. These enzymes levels were at a maximum when the animals were sacrificed 22 to 30 months post-inoculation, a period when the bacterial load in the animals had also reached a maximum. Animals infected with M. leprae but not showing any signs of disseminated infection behaved similar to those in the non-infected group. The observed changes in enzymatic activities were not due to bacterial enzymes and so can be related to tissue damage caused by M. leprae.     

结核分枝杆菌在动物中的流行与传播

结核分枝杆菌(Mycobacterium tuberculosis)是一种重要的人兽共患病病原菌,人类是其主要宿主。千百年来,由于动物与人类关系密切,动物也成为结核分枝杆菌的重要宿主,且感染结核分枝杆菌的动物还能成为传染源,将其传播至人类和其他动物。关于动物感染结核分枝杆菌已有大量报道,包括大象、非人灵长类动物、貘、海狮、犬、猫、牛、鸟等。本文就结核分枝杆菌在野生动物、家畜中的流行与传播进行介绍,总结其在动物间传播的主要途径。     

Experimental inoculation of North American opossums (Didelphis virginiana) with Mycobacterium bovis

Eight North American opossums (Didelphis virginiana) were inoculated with 1 x 10(5) colony forming units of Mycobacterium bovis to investigate their potential as reservoir hosts for bovine tuberculosis in Michigan. Four animals received this dose orally and four were inoculated intramuscularly (i.m.). In each group, two animals were euthanized 1 mo postinoculation (PI) and two at 2 mo PI. Four control animals were housed separately and sacrificed in the same manner as those inoculated. One of four orally inoculated opossums and three of four i.m.-inoculated opossums were positive for M. bovis by culture of tissues obtained at necropsy. The oral recipient had positive cultures from intestine and pooled lymphoid samples. Pooled lymphoid samples were positive in three i.m.-inoculated animals and two of these also had positive liver and lung cultures. One animal with gross and histologic lesions compatible with tuberculosis had negative tissue cultures. The findings suggest that opossums are susceptible to M. bovis infection by multiple routes, although their relative susceptibility compared to true reservoir hosts appears to be low.     

Occurrence, quantification, and genotyping of Mycoplasma conjunctivae in wild Caprinae with and without infectious keratoconjunctivitis

Mycoplasma conjunctivae, the causative agent of infectious keratoconjunctivitis (IKC), was recently detected in asymptomatic Alpine ibex (Capra ibex ibex). This suggested that an external source of infection may not be required for an IKC outbreak in wildlife but might be initiated by healthy carriers, which contradicted previous serologic investigations in chamois. Our aims were to 1) assess the prevalence of M. conjunctivae among asymptomatic ibex and Alpine chamois (Rupicapra rupicapra rupicapra) and its frequency in IKC-affected animals, 2) determine mycoplasma loads in different disease stages, and 3) characterize the M. conjunctivae strains involved. Eye swabs from 654 asymptomatic and 204 symptomatic animals were collected in diverse Swiss regions between 2008 and 2010, and tested by TaqMan real-time PCR. Data analysis was performed considering various patterns of IKC occurrence in the respective sampling regions. Strains from 24 animals were compared by cluster analysis. Prevalence of M. conjunctivae was 5.6% (95% confidence interval [CI]: 3.7-8.1%) in asymptomatic ibex and 5.8% (CI: 3.0-9.9%) in asymptomatic chamois, with significant differences between years and regions in both species. Detection frequency in symptomatic animals was significantly higher during IKC outbreaks than in nonepidemic situations (i.e., regular but low incidence or sporadic occurrence). Mycoplasma load was significantly lower in eyes from healthy carriers and animals with mild signs than from animals with moderate and severe signs. Although some strains were found in both asymptomatic and diseased animals of the same species, others apparently differed in their pathogenic potential depending on the infected species. Overall, we found a widespread occurrence of M. conjunctivae in wild Caprinae with and without IKC signs. Our results confirm the central role of M. conjunctivae in outbreaks but suggest that other infectious agents may be involved in IKC cases in nonepidemic situations. Additionally, presence and severity of signs are related to the quantity of M. conjunctivae in the eyes rather than to the strain. We propose that individual or environmental factors influence the clinical expression of the disease and that persistence of M. conjunctivae in populations of wild Caprinae cannot be excluded.