Genomics and pathogenesis of the avian coronavirus infectious bronchitis virus.

Infectious bronchitis virus (IBV) is a member of the family Coronaviridae, together with viruses such as SARS-CoV, MERS-CoV and SARS-CoV-2 (the causative agent of the COVID-19 global pandemic). In this family of viruses, interspecies transmission has been reported, so understanding their pathobiology could lead to a better understanding of the emergence of new serotypes. IBV possesses a single-stranded, non-segmented RNA genome about 27.6 kb in length that encodes several non-structural and structural proteins. Most functions of these proteins have been confirmed in IBV, but some other proposed functions have been based on research conducted on other members of the family Coronaviridae. IBV has variable tissue tropism depending on the strain, and can affect the respiratory, reproductive, or urinary tracts; however, IBV can also replicate in other organs. Additionally, the pathogenicity of IBV is also variable, with some strains causing only mild clinical signs, while infection with others results in high mortality rates in chickens. This paper extensively and comprehensibly reviews general aspects of coronaviruses and, more specifically, IBV, with emphasis on protein functions and pathogenesis. The pathogenicity of the Australian strains of IBV is also reviewed, describing the variability between the different groups of strains, from the classical to the novel and recombinant strains. Reverse genetic systems, cloning and cell culture growth techniques applicable to IBV are also reviewed.

Investigation of systemic isosporosis outbreaks in an aviary of greenfinch (Carduelis chloris) and goldfinch (Carduelis carduelis) and a possible link with local wild sparrows (Passer domesticus).

CASE REPORT: An outbreak of systemic isosporosis caused mortalities in greenfinches (Carduelis chloris) and goldfinches (Carduelis carduelis) kept in an aviary in the western suburbs of Melbourne. The following year, a further outbreak in the same aviary occurred in a different flock of goldfinches. At the time of the second outbreak, dead and sick common sparrows (Passer domesticus) discovered near the aviary were also found to have systemic isosporosis. METHOD: The systemic isosporosis was investigated and described using histopathology, electron microscopy and sequence analysis of the 18s gene. RESULTS: Isospora spp. infecting the greenfinch and the goldfinch caused significant thickening of the duodenal lamina propria. Measurements in the goldfinches showed an inverse correlation coefficient between the thickening of the duodenum and the weightof the birds. Electron microscopy confirmed the presence of Isospora spp. within lymphocytes migrating into the lamina propria of the duodenum. Analysis of the 18s sequence discovered two different gene sequences across the three species of birds that didn't completely match any sequences previously deposited in GenBank. CONCLUSION: Although the sparrows were found to have died from causes other than systemic Isospora, molecular studies of samples from their liver revealed the presence of an Isospora with 18s gene sequence identical to that found in the captive greenfinches.

Avian mycobacteriosis in captive brolgas (Antigone rubicunda).

CASE SERIES: Avian mycobacteriosis is a significant disease of a wide range of bird species worldwide. The most common causative agent, Mycobacterium avium, is reported to also infect a range of mammals, including humans. Of 11 brolgas (Antigone rubicunda) submitted to the University of Melbourne for postmortem examination over a 10-year period, 7 were diagnosed with mycobacteriosis. All were from a wildlife park and kept in permanent enclosures as part of a breeding program. Most of the brolgas with mycobacteriosis were in poor body condition and had widely disseminated granulomas throughout the body, especially within the liver, spleen and gastrointestinal tract. Respiratory tract involvement was common, with all disseminated cases having pulmonary or air sac granulomas. Rare to moderate numbers of acid-fast organisms were detected in granulomas by histological examination. Where examined by appropriate bacteriological examinations, M. avium complex was isolated from affected tissues. CONCLUSION: This case series is the first known report of mycobacteriosis in brolgas and highlights the pathological changes seen. The complications in maintaining an avian mycobacteriosis-free breeding program and in eradication of the disease from an enclosed wildlife environment are discussed.

High-resolution melt curve analysis to confirm the presence of co-circulating isolates of avian nephritis virus in commercial chicken flocks.

Avian Nephritis Virus (ANV) has been implicated in poor growth and renal disease of young chickens. This paper describes the development of a reverse-transcriptase polymerase chain reaction for the detection of ANV in commercial meat chickens and the use of high-resolution melt curves to detect the presence of genetically different ANVs. Pooled cloacal swabs from both healthy and ill commercial chicken broiler flocks were tested for the presence of ANV using a combination of polymerase chain reaction, molecular cloning, high-resolution melt curve analysis and sequencing. Except for one, all specimens were found to contain two genetically different ANVs. Phylogenetic analysis of the capsid amino acid sequences revealed the presence of four of six groups of ANV identified previously in other countries as well as in two novel groups of ANV. Phylogenetic analysis of nucleotide sequences of partial polymerase, capsid and 3' untranslated regions reveal that the genes of individual ANV virus isolates have different ancestors. This was shown to be due to a template-switching event in the capsid gene that resulted in the 3' end of the capsid gene and the 3' untranslated region of one ANV isolate being transferred to another ANV. These results reveal that infection of chicken flocks with multiple ANV isolates is common and this needs to be taken into consideration in diagnosis of ANV using molecular techniques and in future epidemiological investigations.

Survey of captive parrot populations around Port Phillip Bay, Victoria, Australia, for psittacine beak and feather disease virus, avian polyomavirus and psittacine adenovirus.

OBJECTIVE: This study investigated the prevalence of psittacine beak and feather disease virus (BFDV), avian polyomavirus (APV) and psittacine adenovirus (PsAdV) in captive psittacine birds around Port Phillip Bay, Victoria, Australia. METHODS: Samples of fresh droppings were collected from 118 psittacine birds (109 clinically normal and 9 with feather abnormalities) from 11 avaries in different locations and were used for detection of BFDV, APV and PsAdV using PCR. RESULTS: BFDV, APV and PsAdV were detected in 31%, 13% and 4%, respectively, of the specimens tested. One budgerigar was found to be co-infected with BFDV and PsAdV. At least one sample tested positive for BFDV at each location. CONCLUSION: This is the first report of the prevalence of BFDV, APV and PsAdV in Victoria and provides a foundation for future studies examining the influence of these viruses on the health of aviary birds in Victoria.

The C-terminal end of the capsid protein of Avian Nephritis Virus is antigenic and induces broadly cross-reactive antibodies.

Avian nephritis virus (ANV) has been isolated frequently from commercial broilers in many countries. The prevalence and economic impact of ANV however has been difficult to ascertain due to the lack of convenient serological techniques. In this study the full-length and fragments of the ANV capsid protein were expressed in Baculovirus and affinity purified recombinant proteins used for the detection of ANV antibodies in ELISA. The crystal structure of Human Astrovirus (HAstV) was used as a model to determine potential homologous C-terminal antigenic regions in ANV. The rp37 fragment from three ANV strains NSW_3, ANV-1 and ANV-2, and a shorter NSW_3 fragment (rp33) were compared for their ability to detect ANV antibodies in seven reference chicken sera. The ANV-1 rp37 antigen was the most strain specific whereas the NSW_3 rp37 and rp33 antigens detected antibodies in all heterologous sera, including ANV-1 serum. Irrespective of the strain used, the two NSW_3 protein fragments rp37 and rp33 were found to be superior as antigens for ELISA when compared to the full-length capsid protein rp75. An ELISA designed using the NSW_3 rp33 could reliably differentiate between uninfected and infected commercial broiler flocks, as demonstrated by statistically significant differences between the OD values. This study identified an ANV immunogenic region and successfully used recombinant protein expression of this region to detect cross-reactive ANV antibodies. The results of this study facilitate future studies into the epidemiology and importance of ANV infections in commercial poultry.

Assessment of the potential relationship between egg quality and infectious bronchitis virus infection in Australian layer flocks.

OBJECTIVE: This investigation aimed to determine if there was a relationship between the production of eggs with poor internal quality, as measured by poor Haugh units, by Australian layer flocks and the detection of infectious bronchitis virus (IBV) in the hens. Other risk factors including flock size, flock type, flock age, chicken breed and vaccination frequency were also assessed. METHODS: The study group comprised 17 flocks from 14 farms. Data relating to the factors investigated were requested on a regular basis. The Haugh unit data were used to grade eggs as good or poor based on the age and flock at the time of data collection. Cloacal swabs were collected from 20 chickens in each flock approximately every 6 weeks. RESULTS: IBV was detected from a majority of the flocks and in 68% of cases the IBV strain detected was an A-vaccine-related field strain. Three variant strains were detected. Detection of IBV in a flock, the farm type and flock size were identified as potential risk factors for the production of eggs with poor Haugh units. CONCLUSION: IBV is prevalent in Australian layer flocks, but infection was primarily subclinical. The results complement previous reports indicating that there are many potential risk factors for the production of eggs with poor Haugh units.

Discrepancy between minimal inhibitory concentration to enrofloxacin and mutations present in the quinolone-resistance determining regions of Mycoplasma gallisepticum field strains.

Molecular characterization of the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV in 93 Mycoplasma gallisepticum field strains isolated in different geographic regions revealed discrepancies between minimal inhibitory concentration values and presence of amino acid substitutions within the QRDRs of GyrA and ParC in 9/93 (10%) strains. This may delimitate applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen.

The central role of lipoproteins in the pathogenesis of mycoplasmoses.

Mycoplasmas are a diverse group of pathogens responsible for disease in a wide range of animal species. In recent years there have been considerable advances in knowledge of the proteins and structures involved in adherence in some mycoplasmas, but understanding of the biochemical functions and roles in virulence of another central feature of mycoplasmas, their lipoproteins, continues to develop. The aim of this review is to examine current knowledge of the roles of lipoproteins in the pathogenicity and the evolution of virulence in those mycoplasmas causing disease in domestic animals. Those lipoproteins that have been characterised have roles in adherence, in transport of nutrients into the mycoplasma cell, and in enzymatic interactions with the host. Furthermore they appear to play a prominent role in both inducing the host immune response to infection and in facilitating evasion of this response, particularly through the generation of dramatic levels of antigenic variation on the cell surface. Recent genomic comparisons of several pathogenic mycoplasmas have identified a further level of interaction between lipoproteins and pathogenicity. In several pathogens large scale horizontal gene transfer between distantly related mycoplasma species has resulted in the acquisition of a large number of genes, including those encoding lipoproteins thought to play a role in virulence, by one mycoplasma from another inhabiting the same host species. The interactions between these horizontally transferred genes, their new mycoplasma host and the animal that it infects may be an important contributing factor in the pathogenesis of some mycoplasmoses.

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis.

OBJECTIVE: Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. METHODS: Twenty-six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high-resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. RESULTS: FAdV-8b and FAdV-11 were identified in 13 cases each. In one case, FAdV-1 was also identified. Cross-neutralisation was observed between the FAdV-11 field strain and the reference FAdV-2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV-8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV-11 field strain had the highest identity to FAdV-11 (93.2%) and FAdV-2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. CONCLUSION: These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species.

Epidemiology of recent outbreaks of infectious laryngotracheitis in poultry in Australia.

OBJECTIVE: Over the past 3 years, numerous outbreaks of infectious laryngotracheitis (ILT) have occurred in poultry in Australia. The objectives of this study were to identify the viral strains involved in the recent outbreaks and to determine possible epidemiological links between these outbreaks. PROCEDURE: A combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of several genes of the ILT virus was used to identify genetic differences in field/vaccine ILT virus isolates. In a previous study, these procedures had demonstrated five classes (1-5) in Australia. RESULTS: Analysis of 92 field ILT viruses demonstrated four new classes: 6, 7, 8 and 9. Class 6 was responsible for four outbreaks in one Victorian broiler company and demonstrated to be distinct from other Australian strains of ILT. Class 7 was the Nobilis ILT vaccine (Intervet Pty Ltd). Class 8 was responsible for the majority of the outbreaks in New South Wales and was phylogenetically close to class 7. On one occasion, classes 7 and 8 were identified in an outbreak on a Victorian farm that had used the Nobilis ILT vaccine. Class 9, also phylogenetically close to classes 7 and 8, was found only in New South Wales. The previously identified class 2 was also found to be responsible for a large number of outbreaks, mainly in Victoria. CONCLUSION: The results demonstrate that, epidemiologically, most outbreaks of ILT in New South Wales are unrelated to those in Victoria and suggest a link between classes 8 and 9 and the Nobilis ILT vaccine (class 7).

Application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens.

OBJECTIVE: A real-time polymerase chain reaction (PCR)/high-resolution melt (HRM) curve analysis protocol was developed in our laboratory to differentiate infectious bronchitis (IB) virus reference strains. In the current study, this method was used to detect and classify IB viruses in field submissions. PROCEDURE: Over an 11-month period samples from 40 cases of suspected IB virus were received and 17 submissions were positive for IB virus by polymerase chain reaction. HRM curve analysis classified each strain as subgroup 1, 2 or 3 strain (12 submissions) or a strain that was unable to be classified (5 submissions). The 3' untranslated region (UTR) and partial S1 gene nucleotide sequences for the 17 IB virus strains were determined and their identity with those of the relative reference strains compared to confirm the classifications generated using the HRM curve analysis. RESULTS: Of the 12 IB field viruses classified as subgroup 1, 2, or 3 using HRM curve analysis, the 3'UTR and S1 gene nucleotide sequences had identities ≥99% with the respective subgroup reference strain. Analysis of the 3' UTR and S1 gene nucleotide sequences for the five IB virus strains that could not be classified indicated that four belonged to one of the subgroups, and one was a potential recombinant strain (between strains from subgroups 2 and 3). A novel recombinant strain was also detected. CONCLUSION: HRM curve analysis can rapidly assign the majority of IB viruses present in field submissions to known subgroups. Importantly, HRM curve analysis also identified variant genotypes that require further investigation.

Identification of Chlamydial species in crocodiles and chickens by PCR-HRM curve analysis.

Recently, a PCR protocol (16SG), targeting 16S rRNA gene coupled with high resolution melt (HRM) curve analysis was developed in our laboratory and shown to reliably detect and identify the seven different Chlamydiaceae spp. In this study, the potential of this method was assessed for detection and differentiation of Chlamydiosis in clinical specimens. Of the total number of 733 specimens from a range of animal species, 219 (30%) were found positive by 16SG PCR. When a sufficient amount of DNA was available (64 submissions), amplicons generated by the 16SG PCR were subjected to HRM curve analysis and results were compared to that of nucleotide sequencing. In all instances, the infecting Chlamydiaceae spp. was genotyped according to the identity of its nucleotide sequence to a reference species. Analysis of the HRM curves and nucleotide sequences from 16SG PCR amplicons also revealed the occurrence of a Chlamydophila-like, a Parachlamydia-like and a variant of Chlamydophila psittaci in chickens. These results reveal the potential of 16SG PCR-HRM curve analysis for rapid and simultaneous detection and identification of Chlamydiaceae spp. in animals and demonstrate the capacity of this system for rapid identification of new Chlamydiaceae spp. in animals during routine diagnostic testings.

Effect of a live Mycoplasma synoviae vaccine on the production of eggshell apex abnormalities induced by a M. synoviae infection preceded by an infection with infectious bronchitis virus D1466.

An experimental study was conducted to assess the effect of a live Mycoplasma synoviae vaccine (Vaxsafe MS; Bioproperties Pty Ltd, Ringwood, Victoria, Australia) on M. synoviae-induced eggshell apex abnormalities (EAA). Four experimental groups of specified-pathogen-free white laying hens were made. All groups were inoculated with infectious bronchitis virus D1466 at 18 weeks of age. One group did not receive further treatment (non-vaccinated non-challenged (NVNC)). Two groups were vaccinated at 14 weeks of age against M. synoviae, and one of these groups was also challenged with an EAA-inducing M. synoviae strain 5 days after infectious bronchitis virus challenge (vaccinated non-challenged (VNC) and vaccinated challenged group (VC), respectively). The fourth group was not vaccinated but was challenged with M. synoviae (non-vaccinated challenged (NVC)). Eggs with EAA eggs were produced only in the NVC and VC groups. However, the proportion of eggs with EAA and the mean daily production of eggs with EAA per chicken was significantly lower (P<0.05) in the VC group (88/741 (11.9%) and 0.09+/-0.01 eggs per hen) compared with the NVC group (148/646 (22.9%) and 0.14+/-0.01 eggs per hen). The mean daily egg production per chicken was significantly lower in the NVC group (0.48+/-0.03 eggs) compared with that of the NVNC group (0.60+/-0.03 eggs), but not significantly different from other groups. The eggshell strength of eggs with EAA (22.8 N) was significantly lower (P<0.05) than non-affected eggs from the other groups (33.7 to 39.5 N). Furthermore, the eggshell strength of non-affected eggs in the NVC group was significantly lower (P<0.05) compared with that of non-affected eggs from the flock of origin (33.7 versus 41.2 N), but not different from the other groups. It can be concluded from the present study that vaccination with a live M. synoviae vaccine reduces the occurrence of M. synoviae-induced EAA significantly.

Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene.

AIM: To design a rapid diagnostic test to differentiate species belonging to the family Chlamydiaceae. METHODS AND RESULTS: Five oligonucleotide sets each targeting various conserved regions of the genome of six species (Chlamydia muridarum, C. suis, C. trachomatis, Chlamydophila felis, Cp. pneumoniae and Cp. psittaci) belonging to the family Chlamydiaceae were tested for their suitability for polymerase chain reaction (PCR) and high resolution melt (HRM) curve analysis to differentiate Chlamydiaceae species. Three of the oligonucleotide sets were able to detect all six reference species used in this study, but only one set (16SG) could clearly differentiate between them by HRM curve analysis. The PCR-HRM curve analysis confidence percentages correlated strongly with the nucleotide sequence identities. Clinical specimens from a number of animal species suspected of chlamydiosis were tested with the newly developed 16SG PCR-HRM curve analysis and sequenced to confirm the infecting species. It was demonstrated that PCR-HRM using the 16SG oligonucleotide set could relate the infecting Chlamydiaceae species to the most similar (based on 16S rRNA gene nucleotide sequence) reference species tested. Although Cp. pecorum was not included initially as a reference species in this assay, inclusion of a field isolate of Cp. pecorum as a reference allowed two koala specimens to be correctly identified. CONCLUSION: PCR-HRM analysis using the oligonucleotide set 16SG is a robust, simple and rapid technique for differentiation of at least the Chlamydiaceae species used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique allowed for the rapid detection and identification of the six Chlamydiaceae reference species and may be useful for identification of uncharacterized Chlamydiaceae species or for use in animal species where occurrence of the disease has not been fully investigated.

Glycoprotein G is a virulence factor in infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) is an alphaherpesvirus that causes acute respiratory disease in chickens. The role of glycoprotein G (gG) in vitro has been investigated in a number of alphaherpesviruses, but the relevance of gG in vivo in the pathogenicity of ILTV or in other alphaherpesviruses is unknown. In this study, gG-deficient mutants of ILTV were generated and inoculated into specific-pathogen-free chickens to assess the role of gG in pathogenicity. In chickens, gG-deficient ILTV reached a similar titre to wild-type (wt) ILTV but was significantly attenuated with respect to induction of clinical signs, effect on weight gain and bird mortality. In addition, an increased tracheal mucosal thickness, reflecting increased inflammatory cell infiltration at the site of infection, was detected in birds inoculated with gG-deficient ILTV compared with birds inoculated with wt ILTV. The reinsertion of gG into gG-deficient ILTV restored the in vivo phenotype of the mutant to that of wt ILTV. Quantitative PCR analysis of the expression of the genes adjacent to gG demonstrated that they were not affected by the deletion of gG and investigations in vitro confirmed that the phenotype of gG-deficient ILTV was consistent with unaltered expression of these adjacent genes. This is the first reported study to demonstrate definitively that gG is a virulence factor in ILTV and that deletion of gG from this alphaherpesvirus genome causes marked attenuation of the virus in its natural host.

Rapid differentiation of current infectious bronchitis virus vaccine strains and field isolates in Australia.

OBJECTIVE: Rapid differentiation of vaccine strains of infectious bronchitis virus (IBV) from wild type strains would enhance investigations of disease outbreaks. This study aimed to develop a reverse transcription-polymerase chain reaction (RT-PCR) assay to differentiate between Australian vaccine strains of IBV and field isolates. PROCEDURE: A fragment of 6.5 kilobases that contains the S, M and N genes was amplified by RT-PCR from ten different IBV strains, including vaccine strains and field isolates, and then sequenced. RESULTS: Comparison of the sequences of these strains revealed a deletion of 58 bases in the 3' untranslated region (UTR) of IBV vaccine strains but not in the field isolates. Two primers were designed to amplify a fragment of the 3' UTR that differed in size between the vaccine strains and field isolates. RT-PCR was performed using these two primers to screen 20 IBV strains, including field isolates and the vaccine strains. All strains were correctly identified as either vaccine strains or field isolates. CONCLUSION: This procedure is a rapid, sensitive and inexpensive method for discrimination between most current Australian vaccine strains and field isolates of IBV.

Poor systemic antibody response after vaccination of commercial broiler breeders with Mycoplasma gallisepticum vaccine ts-11 not associated with susceptibility to challenge.

A live attenuated Mycoplasma gallisepticum vaccine, ts-11, has been used for control of M gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. We investigated whether the low level, or lack, of systemic antibodies in ts-11-vaccinated flocks is correlated with susceptibility to infection after challenge with a virulent M. gallisepticum strain. Birds from 2 separate ts-11-vaccinated commercial flocks with no, or weak, rapid serum agglutination responses (at 11 or 14 wk postvaccination) were randomly selected and subjected to aerosol challenge with either M gallisepticum strain Ap3AS or sterile mycoplasma broth. A group of nonvaccinated specific-pathogen-free chickens at similar age were also exposed to aerosolization with M. gallisepticum strain Ap3AS and used as positive controls. Postmortem examination of the birds, performed 2 wk after challenge, revealed no significant difference in microscopic tracheal lesions or mucosal thicknesses between the ts-11-vaccinated field birds irrespective of their aerosolization treatment. However, both microscopic tracheal lesions and tracheal mucosal thicknesses of nonvaccinated challenged birds were significantly greater than those of ts-11 vaccinates. Hence, broiler breeders vaccinated in the field showed significant protection against virulent M. gallisepticum challenge even when no serum antibody was detected by rapid serum agglutination test. These results reveal that seroconversion detected by rapid serum agglutination test after ts-11 vaccination is not a reliable predictor of protection against M. gallisepticum infection. The possible significance of local antibody response and cell-mediated immunity against M. gallisepticum infection is discussed.

Paradoxical effects of IL-12 in leishmaniasis in the presence and absence of vaccinating antigen.

Protective immunity against Leishmania major requires parasite-specific CD4+T helper cells, the development of which is promoted by interleukin 12 (IL-12). In this study we investigated the use of IL-12 DNA to enhance the protective immunity induced by prophylactic vaccination with the L. major Parasite Surface Antigen 2 (PSA-2) DNA. A plasmid was constructed in which the two murine IL-12 subunits p35 and p40 were secreted as a biologically active single chain cytokine. The immunomodulatory effects of this IL-12 DNA were examined by codelivery with PSA-2 DNA in susceptible BALB/c and resistant C3H/He mice and subsequent infection with L. major promastigotes. Surprisingly, administration of IL-12 DNA alone had a protective effect, while coadministration of IL-12 with PSA-2 DNA abrogated protection. This effect of IL-12 DNA was dose dependent and affected by the timing of administration in relation to PSA-2 DNA. The effect of IL-12 on protection was associated with a reduced number of INF-gamma-producing T cells early in infection. A further understanding of this paradoxical effect of IL-12 and possibly other cytokines on protective immunity may be important for their use as adjuvants for Leishmania DNA vaccines.