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Since murine cytomegalovirus (MCMV) was first described in 1954, it has been used to model human cytomegalovirus (HCMV) diseases. MCMV is a natural pathogen of mice that is present in wild mice populations and has been associated with diseases such as myocarditis. The species-specific nature of HCMV restricts most research to cell culture-based studies or to the investigation of non-invasive clinical samples, which may not be ideal for the study of disseminated disease. Initial MCMV research used a salivary gland-propagated virus administered via different routes of inoculation into a variety of mouse strains. This revealed that the genetic background of the laboratory mice affected the severity of disease and altered the extent of subsequent pathology. The advent of genetically modified mice and viruses has allowed new aspects of disease to be modeled and the opportunistic nature of HCMV infection to be confirmed. This review describes the different ways that MCMV has been used to model HCMV diseases and explores the continuing difficulty faced by researchers attempting to model HCMV congenital cytomegalovirus disease using the mouse model.

Introduction: S. aureus is one of the most prevalent potential pathogenic bacteria found in DHM. Some strains produce heat stable enterotoxins that are able to survive pasteurization. Thes...

Background: Some strains of Staphylococcus aureus can produce heat-stable enterotoxins that have been associated with gastritis and potentially necrotizing enterocolitis in preterm infants. Objectives/Hypothesis: To assess the impact of different storage temperatures on S. aureus growth and enterotoxin production in raw, Holder-pasteurized (HP) and ultraviolet-C (UV-C)-treated donated human milk (DHM). Materials and Methods: The milk samples from individual donors were pooled and divided into four equal portions. One portion was HP, the second was UV-C treated, the third was not treated, and the fourth was UV-C treated after being spiked with S. aureus. All samples were incubated at 37°C (18 hours) and 4°C (14 days). Bacterial colony count, enterotoxin A and B, and immune proteins were quantified. Results: At 37°C, the colony count increased in HP DHM and decreased in raw and UV-C-treated DHM. At 4°C, colony counts in HP DHM reduced and were not detected in raw and UV-C-treated DHM from day 8 of incubation. No bacteria were detected in samples that were inoculated before UV-C treatment. Enterotoxin A was only detected in HP-DHM at 37°C from the 9th hour onward. Enterotoxin B was detected in one sample at the 15th hour. Immune protein concentrations were similar in raw and UV-C DHM, and were reduced in the HP DHM. Conclusion: UV-C-treated milk reduces S. aureus growth with similar kinetics to raw milk making it a promising emerging technique to eliminate bacteria while retaining essential immune proteins in DHM.

Preterm infants whose mothers are unable to produce sufficient breast milk are increasingly being supplemented with pasteurised donor human milk (PDHM) instead of commercial preterm infant formula. Concerns have been raised that this practice can result in reduced growth. This retrospective clinical audit collected data from the medical records of a cohort of preterm infants (≤30 weeks gestational age) receiving either ≥28 d of PDHM (n 53) or ≥28 d of their mother’s own milk (MOM, n 43) with standard fortification supplied to both groups during admission. Weight growth velocity was assessed from regained birth weight to 34+1 weeks’ postmenstrual age (PMA); and weight, length and head circumference were compared at discharge and 12 months (corrected age). At 34+1 weeks’ PMA, the weight growth velocity (g/kg per d) was significantly lower in the PDHM group (15·4 g/kg per d, 95 % CI 14·6, 16·1) compared with the MOM group (16·9 g/kg per d, 95 % CI 16·1, 17·7, P=0·007). However, the increase was still within clinically acceptable limits (>15 g/kg per d) and no significant difference was observed in the weight between the two groups. There was no significant difference in weight between the groups at discharge or at the 12-month corrected gestational age review. Although we demonstrated a significant reduction in the weight growth velocity of preterm infants receiving PDHM at 34 weeks’ PMA, this difference is not present at discharge, suggesting that the growth deficit is reduced by supplementation before discharge.

Introduction The bacterial content of donated human milk is either endogenous or introduced via contamination. Defining milk bank bacterial content will allow researchers to devise appropriate tests for significant and commonly encountered organisms. Objective A retrospective audit was conducted on data recorded from the Perron Rotary Express Milk Bank, King Edward Memorial Hospital, Subiaco, Western Australia. This aimed to describe the incidence of bacterial species detected in donated human milk and to identify potentially pathogenic bacteria. Material and methods The data comprised of 2890 batches donated by 448 women between 2007 and 2011. Results Coagulase negative Staphylococcus (CoNS) represented the highest prevalence of bacteria in donated milk, isolated from 85.5% of batches (range: 20 to 650,000CFU/mL) followed by Acinetobacter species in 8.1% of batches (range: 100 to 180,000CFU/mL). Staphylococcus aureus was the most prevalent potentially pathogenic bacteria in 5% of batches (range: 40 to 100,000CFU/mL). Conclusion Further investigation is warranted to better define the risks posed by the presence of toxin-producing S. aureus in raw and pasteurized human milk which may allow minimization of risk to the preterm infants.

Human cytomegalovirus (CMV) is often transmitted through saliva. The salivary gland is a site of CMV replication and saliva can be used to diagnose congenital CMV infections. CMV replication is monitored in whole blood or plasma in renal transplant recipients (RTR) and associates with clinical disease. However, these assays may not detect replication in the salivary gland and there is little data linking detection in saliva with systemic infection and clinical sequelae. RTR (n = 82) were recruited &gt, 2 years after transplantation. An in-house quantitative PCR assay was used to detect CMV UL54 in saliva samples. CMV DNA was sought in plasma using a commercial assay. Vascular health was predicted using flow mediated dilatation (FMD) and plasma biomarkers. CMV-reactive antibodies were quantified by ELISA and circulating CMV-specific T-cells by an interferon-&gamma, ELISpot assay. V&delta, 2&minus, &gamma, &delta, T-cells were detected using multicolor flow cytometry reflecting population expansion after CMV infection. The presence of CMV DNA in saliva and plasma associated with plasma levels of antibodies reactive with CMV gB and with populations of circulating V&delta, T -cells (p &lt, 0.01). T-cells reactive to CMV immediate early (IE)-1 protein were generally lower in patients with CMV DNA in saliva or plasma, but the level of significance varied (p = 0.02&ndash, 0.16). Additionally, CMV DNA in saliva or plasma associated weakly with impaired FMD (p = 0.06&ndash, 0.09). The data suggest that CMV detected in saliva reflects systemic infections in adult RTR.

Preterm infants whose mothers are unable to produce sufficient breast milk are increasingly being supplemented with pasteurised donor human milk (PDHM) instead of commercial preterm infant formula. Concerns have been raised that this practice can result in reduced growth. This retrospective clinical audit collected data from the medical records of a cohort of preterm infants (≤30 weeks gestational age) receiving either ≥28 d of PDHM (n 53) or ≥28 d of their mother's own milk (MOM, n 43) with standard fortification supplied to both groups during admission. Weight growth velocity was assessed from regained birth weight to 34+1 weeks' postmenstrual age (PMA); and weight, length and head circumference were compared at discharge and 12 months (corrected age). At 34+1 weeks' PMA, the weight growth velocity (g/kg per d) was significantly lower in the PDHM group (15·4 g/kg per d, 95 % CI 14·6, 16·1) compared with the MOM group (16·9 g/kg per d, 95 % CI 16·1, 17·7, P=0·007). However, the increase was still within clinically acceptable limits (15 g/kg per d) and no significant difference was observed in the weight between the two groups. There was no significant difference in weight between the groups at discharge or at the 12-month corrected gestational age review. Although we demonstrated a significant reduction in the weight growth velocity of preterm infants receiving PDHM at 34 weeks' PMA, this difference is not present at discharge, suggesting that the growth deficit is reduced by supplementation before discharge.

© FEMS 2018. All rights reserved. The American Society for Microbiology's curricular guidelines for Introductory Microbiology highlighted key laboratory skills in the isolation, visualization and identification of microorganisms as core learning objectives in the discipline. Since the publication of these guidelines in 2012, there has been a paucity of diagnostic assessment tools in the literature that can be used to assess competencies in the microbiology laboratory. This project aimed to establish a laboratory competency examination for introductory microbiology, with tasks specifically aligned to laboratory skills and learning outcomes outlined in curricular guidelines for microbiology. A Laboratory Competency Examination assessing student skills in light microscopy, Gram-staining, pure culture, aseptic technique, serial dilution, dilution calculations and pipetting was developed at The University of Queensland, Australia. The Laboratory Competency Examination was field-tested in a large introductory microbiology subject (~400 students), and student performance and learning gains data were collected from 2016 to 2017 to evaluate the validity of the assessment. The resulting laboratory assessment is presented as an endpoint diagnostic tool for assessing laboratory competency that can be readily adapted towards different educational contexts.

There is a paucity of data on the effect of preterm birth on the immunological composition of breast milk throughout the different stages of lactation. We aimed to characterise the effects of preterm birth on the levels of immune factors in milk during the 1st month postpartum, to determine whether preterm milk is deficient in antimicrobial factors. Colostrum (days 2–5 postpartum), transitional milk (days 8–12) and mature milk (days 26–30) were collected from mothers of extremely preterm (n15), very preterm (28–n15), moderately preterm (32–n15) and term infants (37–41 weeks of gestation,n15). Total protein, lactoferrin, secretory IgA, soluble CD14 receptor (sCD14), transforming growth factor-β2 (TGF-β2),αdefensin 5 (HD5),βdefensins 1 (HBD1) and 2, IL-6, IL-10, IL-13, interferon-γ, TNF-αand lysozyme (LZ) were quantified in milk. We examined the effects of lactation stage, gestational age, volume of milk expressed, mode of delivery, parity and maternal infection on milk immune factor concentrations using repeated-measures regression analysis. The concentrations of all factors except LZ and HD5 decreased over the 1st month postpartum. Extremely preterm mothers had significantly higher concentrations of HBD1 and TGF-β2 in colostrum than term mothers did. After controlling for other variables in regression analyses, preterm birth was associated with higher concentrations of HBD1, LZ and sCD14 in milk samples. In conclusion, preterm breast milk contains significantly higher concentrations of some immune proteins than term breast milk.

Pasteurized donor human milk is provided by milk banks to very preterm babies where their maternal supply is insufficient or unavailable. Donor milk is currently processed by Holder pasteurization, producing a microbiologically safe product but significantly reducing immunoprotective components. Ultraviolet-C (UV-C) irradiation at 254 nm is being investigated as an alternative treatment method and has been shown to preserve components such as lactoferrin, lysozyme and secretory IgA considerably better than Holder pasteurization. We describe the inactivation of cytomegalovirus, a virus commonly excreted into breast milk, using UV-C irradiation. Full replication was ablated by various treatment doses. However, evidence of viral immediate early proteins within the cells was never completely eliminated indicating that some viral gene transcription was still occurring. In conclusion, UV-C may be a safe alternative to pasteurisation for the treatment of human donor milk that preserves the bioactivity. However, our data suggests that CMV inactivation will have to be carefully evaluated for each device designed to treat breast milk using UV-C irradiation.

Antibodies reactive with the ovarian glycoprotein zona pellucida (ZP) have been linked with human female infertility. Anti-fertility vaccines that target ZP antigens have been utilized to restrict pest animal populations and their efficacy is associated with ovary-specific antibody induction. However, the necessity for zona pellucida-specific antibody in mediating infertility has not been examined in vivo. A recombinant mouse cytomegalovirus vaccine encoding murine zona pellucida 3 that induces rapid and complete infertility in BALB/c mice has been produced. The onset of infertility is temporally related to the presence of antibody sequestered into ovarian follicles and binding to the ZP of infected mice and the loss of mature follicles. When this vaccine was inoculated into immunoglobulin-deficient BALB/c mice with a null mutation in the immunoglobulin mu chain gene Igh-6, fertility was unaffected. Passive transfer of serum containing ZP3 antibodies also elicited transient infertility. Electron microscopy of ovarian tissue collected from ZP3-immunized immunocompetent mice demonstrated significant focal thinning of the zona pellucida (ZP) with reduced length and concentration of transzonal processes and many oocytes displayed evidence of injury. None of these changes were found in vaccinated immunoglobulin-deficient mice. These data confirm that ZP3-reactive antibody is necessary and sufficient to induce autoimmune-mediated follicular depletion and fertility suppression following the inoculation of this vaccine, and suggest that this is due to impaired zona pellucida formation. These findings have relevance in understanding the etiology of autoimmune ovarian disease in woman where anti-ZP antibodies are likely to have a causal role in infertility.

The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as a vaccine expressing murine zona pellucida 3 (mZP3) for viral vectored immunocontraception (VVIC) in mice. However, certain laboratory strains of mice are resistant to infection with K181 and therefore demonstrate resistance to VVIC. Cmv1 is the best characterised innate resistance mechanism to MCMV and was first described in C57BL/6 mice. Resistance in C57BL/6 mice is due to early and strong activation of natural killer (NK) cells by an MCMV gene product, m157, that binds directly to the NK cell activating receptor Ly49H. In this study a wild strain of MCMV, G4, which expresses a variant m157 incapable of activating Ly49H, was engineered to express murine zona pellucida 3 (mZP3) and assessed for its ability to sterilise female C57BL/6 mice. When infected with K181-mZP3 female C57BL/6 mice remained fully fertile. In contrast, female C57BL/6 mice were sterilised by a single intraperitoneal inoculation of G4-mZP3. Infertility was induced by G4-mZP3 in three strains of mice that express Ly49H, on two different histocompatibility-2 (H-2) backgrounds. Finally, enhanced immunocontraception was observed in mice expressing H-2(k) mediated resistance to MCMV when infected with G4-mZP3 compared to K181-mZP3. These data indicate that when using viral vaccine vectors, variant vector strains may be used to circumvent powerful innate immune responses against the vector and promote effective vaccination. This study highlights the importance of vaccine vector genetics in vaccination strategies.

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4(+) and CD8(+) T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immunocontraception (VVIC). MCMV expressing murine zona pellucida 3 (mZP3) induces long term infertility in up to 100% of female BALB/c mice following a single inoculation. Whilst a large number of antigens have been investigated as potential immunocontraceptive vaccines, it has been difficult to compare these antigens as few studies have used identical approaches or even animal species. Here a range of protein and polyepitope antigens, all expressed by MCMV, were tested for the ability to sterilise female mice. The antigens tested were bone morphogenic protein 15 (BMP15), oviduct glycoprotein (OGP) and ubiquitin-tagged mZP3. In addition, four polyepitope constructs that contain rodent or mouse specific epitopes were tested. This study found that when expressed by an MCMV vector, only full-length mZP3 or ubiquitin-tagged mZP3 induced infertility in female mice. BMP15 and OGP had no effect. Of the four polyepitopes tested, one had a partial effect on fertility. These data indicate that while MCMV is an effective vector for VVIC, the antigen used needs to be tested empirically. The partial infertility seen in mice infected with one of the polyepitope vaccines is a promising finding suggesting that it may be possible to combine a species specific virus with a species specific antigen for use as a disseminating mouse control agent.

Background Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. Methods Sixty mothers of extremely preterm (

As with human cytomegalovirus (HCMV) infection of humans, murine CMV (MCMV) infection is widespread in its natural host, the house mouse Mus domesticus, and may consist of mixed infection with different CMV isolates. The incidence and mechanisms by which mixed infection occurs in free-living mice are unknown. This study used two approaches to determine whether mixed infection with MCMV could be established in laboratory mice. The first utilized two naturally occurring MCMV strains, N1 and G4, into which the lacZ gene was inserted by homologous recombination. The lacZ gene was used to track recombinant and parental viruses in simultaneously coinfected mice. In the second approach, a real-time quantitative PCR (qPCR) assay was used to detect viral immediate-early 1 (ie1) gene sequences in mice successively coinfected with G4 and then with the K181 MCMV strain. In both systems, mixed infection was detected in the salivary glands and lungs of experimentally infected mice. MCMV-specific antibody in sera and G4 IE1-specific cytotoxic lymphocyte responses in the spleens of twice-infected mice did not prevent reinfection. Finally, the prevalence of mixed infection in free-living mice trapped in four Australian locations was investigated using real-time qPCR to detect ie1 DNA sequences of N1, G4 and K181. Mixed infection with MCMVs containing the G4 and K181 ie1 sequences was detected in the salivary glands of 34·2 % of trapped mice. The observations that mixed infections are common in free-living M. domesticus and are acquired by immunocompetent mice through simultaneous or successive infections are important for vaccine development.

Mouse PH20 (mPH20), the mouse homologue to guinea pig hyaluronidase protein PH20 (gpPH20), was used to produce contraceptive vaccines that target both sexes of mice. Previously, immunization with a female gamete antigen (the zona pellucida subunit 3 protein) delivered in a recombinant murine cytomegalovirus (MCMV), or as a purified recombinant protein, has been shown to induce infertility in female mice. There is evidence, however, that sperm protein antigens could provide broader contraceptive coverage by affecting both males and females, and the most promising has been gpPH20 when tested in a guinea pig model. Mice were therefore either inoculated with a recombinant MCMV expressing mPH20 or immunized directly with purified recombinant mPH20 protein fused to maltose-binding protein. Mice treated with either vaccine formulation developed serum antibodies that cross-reacted to a protein band of 55 kDa corresponding to mPH20 in Western blots of mouse sperm. However, there was no significant reduction in the fertility of males or females compared with control animals with either formulation. We conclude from our data that recombinant mPH20 is not a useful antigen for inclusion in immunocontraceptive vaccines that target mice.

House mice (Mus domesticus) were recently introduced to Thevenard Island, off the northwest coast of Western Australia. This island is also habitat for an endangered native rodent, the short-tailed mouse (Leggadina lakedownensis). Concerns have been raised that house mice may pose a threat to L. lakedownensis both through competition and as a source of infection. To assess the threat to L. lakedownensis posed by viral pathogens from M. domesticus, a serological survey was conducted from 1994 to 1996 of both species for evidence of infection with 14 common murine viruses (mouse hepatitis virus, murine cytomegalovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus strains FL and K87, minute virus of mice, mouse parvovirus, reovirus type 3, Sendai virus, Theiler's mouse encephalomyelitis virus, polyoma virus, pneumonia virus of mice, and encephalomyocarditis virus) and Mycoplasma pulmonis. Despite previous evidence that populations of free-living M. domesticus from various locations on the Australian mainland were infected with up to eight viruses, M. domesticus on Thevenard Island were seropositive only to murine cytomegalovirus (MCMV). Antibodies to MCMV were detected in this species at all times of sampling, although seroprevalence varied. Infectious MCMV could be isolated in culture of salivary gland homogenates from seropositive mice. In contrast, L. lakedownensis on Thevenard Island showed no serological evidence of infection with MCMV, any of the other murine viruses, or M. Pulmonis, and no virus could be isolated in culture from salivary gland homogenates. Although MCMV replicated to high titers in experimentally infected inbred BALB/c laboratory mice as expected, it did not replicate in the target organs of experimentally inoculated L. lakedownensis, indicating that the strict host specificity of MCMV may prevent its infection of L. lakedownensis. These results suggest that native mice on Thevenard Island are not at risk of MCMV infection from introduced house mice, and raise interesting questions about the possible selective survival of MCMV in small isolated populations of M. domesticus.

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Species-specific viruses are being genetically engineered to produce contraceptive biological controls for pest animals such as mice, rabbits and foxes. The virus vaccines are intended to trigger an autoimmune response in the target animals that interferes with their fertility in a process termed virally vectored immunocontraception. Laboratory experiments have shown that high levels of infertility can be induced in mice infected with recombinant murine cytomegalovirus and ectromelia virus expressing reproductive antigens as well as in rabbits using myxoma virus vectors. The strategies used to produce and deliver species-specific immunocontraceptive vaccines to free-living wildlife are presented in this review. Discussion includes coverage of the likely safety of the proposed vaccines as well as the implications of the approach for fertility control in other species.

Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) expressing the mouse egg-coat protein zona pellucida 3 (mZP3) has been shown to sterilise female mice by breaking self-tolerance and inducing an immune response against the host ZP3. This virus has the potential to be used for mouse population control, however the effect of this recombinant immunocontraceptive virus in non-host species must be determined. Recombinant MCMV-mZP3, based on both laboratory and wild strains of virus, induced long-lived antibody responses against structural viral proteins and mZP3 when inoculated into laboratory rats, although no viral DNA or replicating virus was identified. The anti-mZP3 antibodies were specific for mouse ZP3, did not cross-react with rat ZP3, and had no effect on the fertility of the rats.

Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.

The wild house mouse (Mus domesticus) is not native to Australia and was introduced from Europe with early settlement. It undergoes periodic population explosions or plagues, which place significant economic and social burdens on agricultural communities. Present control mechanisms rely on improvements to farm hygiene and the use of rodenticides. This review covers over a decade of work on the use of virally vectored immunocontraception (VVIC) as an adjunct method of controlling mouse populations. Two viral vectors, ectromelia virus (ECTV) and murine cytomegalovirus (MCMV) have been tested as potential VVIC vectors: MCMV has been the most widely studied vector because it is endemic to Australia; ECTV less so because its use would have required the introduction of a new pathogen into the Australian environment. Issues such as efficacy, antigen choice, resistance, transmission, species specificity and safety of VVIC are discussed. In broad terms, both vectors when expressing murine zona pellucida 3 (mZP3) induced long-term infertility in most directly inoculated female mice. Whereas innate and acquired resistance to MCMV may be a barrier to VVIC, the most significant barrier appears to be the attenuation seen in MCMV-based vectors. This attenuation is likely to prevent sufficient transmission for broad-scale use. Should this issue be overcome, VVIC has the potential to contribute to the control of house mouse populations in Australia.

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1). These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control. (1)Lloyd ML, et al. (2003). Biol. Reprod. 68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel. 16(Supplement), 77.

A recombinant mouse virus, murine cytomegalovirus, that has been engineered to encode the fertility antigen murine zona pellucida 3 (mZP3), is being developed for fertility control in mice. A single inoculation of the recombinant virus induces complete infertility in female BALB/c mice which persists for the breeding life of the animal. The extent of this autoimmune response was unexpected especially as the incorporation of mZP3 appears to have immunologically attenuated the growth of the recombinant virus. The histological features of the infection are an initial depletion in tertiary follicles by 21 days post inoculation followed by a progressive depletion of primordial follicles, leading to an almost complete absence of follicles by 150 days post-infection. High titre, long lasting, zona pellucida-specific antibody is present in infertile BALB/c mice although infertility has not been linked with either a critical titre or a dominant immunoglobulin isotype. However, our evidence suggests that anti-ZP3 antibody plays a primary role in infertility since antibodies are detected in vivo bound to the zona pellucida of ovaries from recombinant virus-infected mice, and passively transferred antibody from infected animals induces infertility in the absence of recombinant virus. In addition, an experiment in which immunoglobulin-deficient mice remained fertile after inoculation with the recombinant virus indicates that antibody is crucial for the immunocontraceptive effect to occur. Other immune mechanisms are also being explored.

Immunocontraception is a promising biological control for wild mice in Australia, having the potential to reduce the socioeconomic cost of plagues with minimal environmental impact. Inoculation of BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (mCMV-ZP3) confers total infertility characterised by depletion in ovarian tertiary follicles by Day 21 post inoculation followed by a progressive depletion in primordial follicles (1). The mechanisms underlying ovarian pathology are largely unknown but are likely to involve antibody mediated and cell mediated immune responses. The immune pathology may also be facilitated by acute responses involving antibody binding to ZP in growing follicles resulting in recruitment of inflammatory cells and oocyte destruction. The aim of this study was to investigate the effect of mCMV–ZP3 infection on leukocyte infiltration and expression of oocyte-derived signalling molecules in ovarian tissue. Fifteen BALB/c female mice were randomly allocated into three groups of 5 animals. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2�×�104�p.f.u. of mCMV and mCMV-ZP3 respectively. Ovaries were retrieved at Day 7 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages. The other ovary was processed for real time quantitative RT-PCR analysis of growth and differentiation factor 9 (GDF-9) and connexin 43 (Cx43) expression. mCMV-ZP3 inoculation increased the abundance of ovarian leukocytes (P�=�0.08), significantly increased expression of Cx43 mRNA (p

Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in the host. A recombinant murine cytomegalovirus expressing the mouse ovarian glycoprotein zona pellucida 3 induces long lasting sterility in female BALB/c mice. Using inbred mouse strains selected for their innate resistance or susceptibility to MCMV, we show that genetically determined innate resistance to MCMV can reduce immunocontraceptive success. The Cmv1 locus that controls natural killer cell mediated responses to MCMV was implicated in determining vaccine efficacy. However, the role of the H-2 haplotype was less clear. Interestingly, Mus domesticus from an outbred colony of wild-derived mice were readily sterilised by vaccination, consistent with observations that strong innate immunity to MCMV is not common in Australian wild mice.