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Introduction: Bovine Genital Campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is a sexually transmitted bacterium that significantly impacts cattle reproductive performance. However, current detection methods lack consistency and reliability due to the close genetic similarity between C. fetus subsp. venerealis and C. fetus subsp. fetus. Therefore, this study aimed to utilize complete genome analysis to distinguish genetic features between C. fetus subsp. venerealis and other subspecies, thereby enhancing BGC detection for routine screening and epidemiological studies., Methods and Results: This study reported the complete genomes of four C. fetus subsp. fetus and five C. fetus subsp. venerealis, sequenced using long-read sequencing technologies. Comparative whole-genome analyses ( n = 25) were conducted, incorporating an additional 16 complete C. fetus genomes from the NCBI database, to investigate the genomic differences between these two closely related C. fetus subspecies. Pan-genomic analyses revealed a core genome consisting of 1,561 genes and an accessory pangenome of 1,064 genes between the two C. fetus subspecies. However, no unique predicted genes were identified in either subspecies. Nonetheless, whole-genome single nucleotide polymorphisms (SNPs) analysis identified 289 SNPs unique to one or the C. fetus subspecies. After the removal of SNPs located on putative genomic islands, recombination sites, and those causing synonymous amino acid changes, the remaining 184 SNPs were functionally annotated. Candidate SNPs that were annotated with the KEGG "Peptidoglycan Biosynthesis" pathway were recruited for further analysis due to their potential association with the glycine intolerance characteristic of C. fetus subsp. venerealis and its biovar variant. Verification with 58 annotated C. fetus genomes, both complete and incomplete, from RefSeq, successfully classified these seven SNPs into two groups, aligning with their phenotypic identification as CFF ( Campylobacter fetus subsp. fetus) or CFV/CFVi ( Campylobacter fetus subsp. venerealis and its biovar variant). Furthermore, we demonstrated the application of mraY SNPs for detecting C. fetus subspecies using a quantitative PCR assay., Discussion: Our results highlighted the high genetic stability of C. fetus subspecies. Nevertheless, Campylobacter fetus subsp. venerealis and its biovar variants encoded common SNPs in genes related to glycine intolerance, which differentiates them from C. fetus subsp. fetus. This discovery highlights the potential of employing a multiple-SNP assay for the precise differentiation of C. fetus subspecies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ong, Blackall, Boe-Hansen, deWet, Hayes, Indjein, Korolik, Minchin, Nguyen, Nordin, Siddle, Turni, Venus, Westman, Zhang and Tabor.)

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus , and Streptococcus . All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/ Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

The objective of this study was to confirm the presence of seven species of Eimeria involved in chicken coccidiosis in Australia by comparing internal transcribed spacer 1 (ITS-1) sequences, ITS-1 polymerase chain reaction (PCR) methods and to apply phylogenetic analysis to assess evolutionary relationships of Australian isolates. Twenty-two distinct ITS-1 regions of 15 Australian Eimeria isolates were sequenced, and analysed using maximum parsimony, distance and maximum likelihood methods. Poor bootstrap support, resulting from high ITS-1 sequence heterogeneity between all species groups, resulted in polychotomy of the Eimeria species in all three trees generated by these analyses. Percentage identity analyses revealed two distant ITS-1 lineages in both E. mitis and E. maxima at the same levels that separate the two species E. tenella and E. necatrix. One E. maxima lineage consisted of Australian isolates, the other American isolates, with one European sequence (originating from the same isolate) in each lineage. One Australian E. praecox sequence was only distantly related (33% variation) to three E. praecox sequences from Australian and European isolates. Short and long ITS-1 variants were isolated from both E. tenella (cloned line) and E. necatrix isolates with deletions (106 and 73 bp, respectively) in the short variants within the 3' region of the ITS-1 sequence. ITS-1 sequences of strains of both E. brunetti and E. acervulina species varied the least. Apart from E. maxima, all of the ITS-1 sequences of the six remaining individual species clustered to the exclusion of other species in all phylogenetic trees. Published ITS-1 tests for E. necatrix, E. acervulina, E. brunetti and E. tenella, combined with three new tests for E. mitis, E. praecox and Australian E. maxima amplified all respective Australian isolates specifically in a nested format using conserved ITS-1 PCR products as template to improve the sensitivity. All PCR tests were confirmed against a collection of 24 Australian chicken Eimeria isolates and contaminating species were detected in some instances. In conclusion, once the genetic variation between species and strains is determined, the ITS-1 is a good target for the development of species-specific assays, but the ITS-1 sequences alone do not seem suitable for the confirmation of phylogenetic inferences for these species. This study reports the first attempt at the analysis of the phylogeny and sequence comparison of the Eimeria species involved in chicken coccidiosis in Australia.

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.

Objective: To demonstrate the value of PCR assays to determine the genotypes of Babesia bovis in cattle with clinical signs of babesiosis within 3 weeks after vaccination against tick fever., Design: Samples from 5 cases of babesiosis in cattle soon after vaccination against tick fever were analysed in two PCR assays., Procedure: Parasite DNA was purified from blood taken from cattle with signs of babesiosis within 3 weeks of vaccination against tick fever. DNA was also prepared from the tissues of animals that died of babesiosis. Two PCR assays that amplify repeat sequences of DNA within the B bovis genes, Bv80 and BvVA1, were used to differentiate the genotypes of field isolates and vaccine strains of B bovis., Results: One of the five cases of babesiosis was found to be caused by a vaccine strain, but PCR analyses showed that the predominant isolate in the other four cases was not the vaccine strain., Conclusions: PCR assays on the DNA of B bovis obtained from the blood or tissues of cattle clinically affected with tick fever within 3 weeks after vaccination are useful to distinguish between vaccine strains and field isolates as the source of infection.

Objective: To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed., Design: A comparative study of polymerase chain reaction genotypes in different populations of B bovis., Procedure: Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated., Results: No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype., Conclusion: No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.