The role of the sequential organ failure assessment score in evaluating the outcome in dogs with parvoviral enteritis.

SCIENTIFIC BACKGROUND: The aim of this prospective study was to assess whether the Sequential Organ Failure Assessment (SOFA) score could be indicative of outcome (survival to discharge) in dogs with parvoviral enteritis. METHODS: In 35 naturally infected dogs, the SOFA score and clinical score were calculated and the presence of systemic inflammatory response syndrome was verified on admission and during the first four days of hospitalization. RESULTS: 26 dogs survived, and out of the 9 non-survivors, 6 dogs had positive blood cultures. Mean SOFA scores and clinical scores between survivors and non-survivors and between septic and non-septic dogs on admission and on each hospitalization day were significantly different. Trends in SOFA score indicated that in non-survivors and septic dogs there was an increase in SOFA score during the first four days of hospitalization and a decrease occurred in survivors and non-septic dogs. The area under the curve (ROC curve analysis) for SOFA score predicting the outcome was 0.797 and predicting sepsis was 0.834. The best cut-off point of SOFA score for predicting the final outcome was 3.5 and the best cut-off of SOFA score for predicting sepsis was also 3.5. CONCLUSIONS: Either single values or trends in SOFA score can assist in suspecting sepsis and reaching prognosis in parvoviral enteritis.

Limited cross-species transmission and absence of mutations associated with SARS-CoV-2 adaptation in cats: A case study of infection in a small household setting.

In the present study, the course of SARS-CoV-2 natural infection in two asymptomatic cats, which were negative for immunosuppressive retroviral infections, is investigated. The source of the virus for the cats was their COVID-19-affected owner, with whom they were in continuous proximity in a small household setting. The owner's signs included fatigue, sneezing, anosmia and loss of taste, and diagnosis was confirmed 4 days after symptom onset. Oropharyngeal and faecal swabs were collected from the cats, to investigate the course of SARS-CoV-2 RNA concentrations, as well as the directionality of the chain of virus transmission. Both infected cats were real-time RT-PCR-positive on various time-points. Pharyngeal shedding of at least 6 days was observed in them, with high SARS-CoV-2 titres (> 7 Log10 copies/swab) on the first sampling time-point, that is, 7 days after the onset of owner's clinical signs. In one cat, after the initial decline, slightly increasing virus titres were measured 3 to 6 days after the first real-time RT-PCR-positive swab. Serological testing of this cat revealed absence of seroconversion. The course of viral RNA concentrations in the faecal swabs of the other cat was similar to that in its pharynx. The detected SARS-CoV-2 strains, from both infected cats and their owner, underwent whole-genome sequencing, revealing the absence of emergence of cross-species adaptive mutations in cats. The results support the notion that human SARS-CoV-2 strains are relatively well-adapted to cats. It is still unclear whether asymptomatic animals could play a role in COVID-19 epidemiology, in case of interaction with naïve animals and/or people. Our findings highlight difficulties in SARS-CoV-2 transmission to cats, as neither the two infected cats nor their owner was able to transmit the virus to a third cat living in the same small flat, despite their very close contact during the days corresponding to high virus shedding.

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination.

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3-1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

A one-step real-time RT-PCR assay for simultaneous typing of SARS-CoV-2 mutations associated with the E484K and N501Y spike protein amino-acid substitutions.

The emergence of SARS-CoV-2 mutations resulting in the S protein amino-acid substitutions N501Y and E484K, which have been associated with enhanced transmissibility and immune escape, respectively, necessitates immediate actions, for which their rapid identification is crucial. For the simultaneous typing of both of these mutations of concern (MOCs), a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed. The assay is highly sensitive with a LOD of 117 copies/reaction, amplification efficiencies >94 % and a linear range of over 5 log10 copies/reaction. Validation of the assay using known SARS-CoV-2-positive and negative samples from human and animals revealed its ability to correctly identify wild type strains, and strains possessing either one or both targeted amino-acid substitutions, thus comprising a useful pre-screening tool for rapid MOC identification. The basic principles of the methodology for the development of the assay are explained in order to facilitate the rapid design of similar assays able to detect emerging MOCs.

Prevalence and mechanisms of resistance to fluoroquinolones in Pseudomonas aeruginosa and Escherichia coli isolates recovered from dogs suffering from otitis in Greece.

The aim of this study was to investigate the prevalence and the implicated mechanisms of resistance against selected veterinary fluoroquinolones (enrofloxacin, marbofloxacin and pradofloxacin) among 101 Pseudomonas aeruginosa (n=75) and Escherichia coli (n=26) isolates collected from dogs suffering from otitis. Resistance ranged from 32.0% to 48.0% with differences not being considered statistically significant among the three agents or between the two bacterial species. However, individual MICs of pradofloxacin, the latest veterinary fluoroquinolone, were significantly lower than those of enrofloxacin, the oldest one, indicating an increased in vitro potency of the former antimicrobial. Pradofloxacin MIC90 was, additionally, the lowest (8µg/ml), in E. coli, or among the lowest (8µg/ml), in P. aeruginosa isolates. Resistance was in most cases associated with topoisomerase substitutions, with patterns GyrA:V73G in P. aeruginosa and GyrA:S83L+D87N/ParC:S58I+A86V in E. coli being reported for the first time in small animal isolates. Only 6.7% and 15.4% of P. aeruginosa and E. coli otitis isolates, respectively, carried plasmid-mediated quinolone resistance (PMQR) genes, which, moreover, contributed minimally to resistance. Efflux pump activity was additionally detected in resistant E. coli isolates, even those lacking topoisomerase substitutions or PMQR genes. The emergence of resistance in the canine otitis isolates seemed to be associated with previous, prolonged systemic fluoroquinolone administration. In any case, antimicrobial susceptibility testing should guide the selection of systemic FQs for the treatment of canine otitis.

Pharmacodynamic interactions of amikacin with selected ß-lactams and fluoroquinolones against canine Escherichia coli isolates.

Knowledge of in vitro antimicrobial interactions can serve as a guide for clinical application of combination antimicrobial regimens. The aim of the present study was to determine the pharmacodynamic interactions of amikacin with either amoxicillin/clavulanic acid, ceftazidime, enrofloxacin or marbofloxacin against clinical canine Escherichia coli isolates. Bactericidal activity of individual antimicrobials was assessed by use of static kill curves. Interactions between amikacin and each of the ß-lactams or fluoroquinolones were subsequently analyzed by employing the fractional maximal effect method. Amikacin, compared with all other agents, displayed the most rapid and extensive bacterial killing, the lowest level (with respect to MIC) at which half the maximal effect was observed and the most linear concentration-effect relationship. The combinations of amikacin with amoxicillin/clavulanic acid or ceftazidime were completely synergistic in four and three out of the five investigated isolates, respectively, with additivity being sporadically observed. On the other hand, the combinations of amikacin with enrofloxacin or marbofloxacin yielded a mosaic of interaction types with no discernible pattern or differentiation between fluoroquinolone-susceptible and resistant isolates; synergy was only infrequently observed, mainly at increased fluoroquinolone concentrations. In conclusion, the combinations of amikacin with the two ß-lactams were found to be more promising, in terms of synergy achievement, compared with the respective combinations with the two fluoroquinolones.

Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections.

Canine tick-borne pathogens such as Ehrlichia canis and Hepatozoon canis are widespread in the Mediterranean basin but have never been reported or investigated in Cyprus. We describe herein the presence of canine tick-borne pathogens in three dogs with clinical signs compatible with vector-borne diseases from Paphos area of Cyprus. Molecular and phylogenetic analysis revealed the presence of E. canis, Anaplasma platys, H. canis, Babesia vogeli and Mycoplasma haemocanis in Cyprus. One dog co-infected with E. canis, H. canis, B. vogeli and M. haemocanis is, to the best of our knowledge, the first report of this multiple co-infection in dogs. The tick-borne pathogens reported in the current study should be considered in the differential diagnoses in dogs exposed to ticks in Cyprus.

Cardiac troponin I concentrations, electrocardiographic and echocardiographic variables remained unchanged in dogs experimentally infected with Ehrlichia canis.

Canine monocytic ehrlichiosis (CME, Ehrlichia canis) has occasionally been associated with myocardial injury. The aim of the present study was to serially measure and evaluate cardiac troponin I (cTnI) concentrations in dogs with experimentally induced acute and subclinical CME and to evaluate potential associations between cTnI concentration and an array of echocardiographic and electrocardiographic parameters. Serum cTnI concentration and simultaneous echocardiographic and electrocardiographic recordings were evaluated in 12 healthy Beagle dogs prior to experimental infection and on days 20 and 90 post-inoculation with E. canis. Almost all serum cTnI concentrations were below the limit of detection and selected electrocardiographic and echocardiographic parameters remained unchanged throughout the study.

Acute phase protein and antioxidant responses in dogs with experimental acute monocytic ehrlichiosis treated with rifampicin.

There is currently lack of information on the changes of acute phase proteins (APP) and antioxidant markers and their clinical relevance as treatment response indicators in canine monocytic ehrlichiosis (CME). The objective of this study was to investigate the patterns of C-reactive protein (CRP), haptoglobin (Hp), ferritin and paraoxonase-1 (PON-1) during treatment of dogs with acute CME with rifampicin. Blood serum samples from ten Beagle dogs with experimental acute CME were retrospectively examined. Five dogs (Group A) were treated with rifampicin (10mg/Kg/24h), per os, for 3 weeks and 5 dogs (Group B) received no treatment (infected controls). Two Beagle dogs served as uninfected controls. Blood serum samples were serially examined prior to Ehrlichia canis inoculation and on post-inoculation days 14, 21, 28, 35 and 42. Significant changes of CRP, Hp, ferritin and PON-1 values were found in the majority of infected dogs. However, their concentrations did not differ between the two groups during the treatment observation period. The results of this study indicate that although several APP and PON-1 tend to significantly change in the majority of dogs with acute CME, they were of limited clinical relevance as treatment response indicators in this experimental setting.

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.

Synovial fluid cytology in experimental acute canine monocytic ehrlichiosis (Ehrlichia canis).

Evidence-based information of a cause-and-effect relationship between Ehrlichia canis infection and polyarthritis in naturally- or experimentally-infected dogs is currently lacking. The aim of this prospective study was to investigate whether synovial fluid cytological evidence of arthritis could be documented in dogs with acute monocytic ehrlichiosis. Direct synovial fluid cytology smears from eight Beagle dogs experimentally infected with E. canis were examined prior to, and on 21, 35 and 63 days post-inoculation. The cytological variables assessed included cellularity, percentages of mononuclear cells and neutrophils, macrophage reactivity and evidence of E. canis morulae. The median cellularity and percentages of mononuclear cells and neutrophils prior to inoculation did not differ when compared to post-inoculation cytological evaluation. Increased cellularity, E. canis morulae or cytological evidence of arthritis or macrophage reactivity were not observed throughout the course of the study. In the present study, no cytological evidence of arthritis was found in dogs with experimental acute canine monocytic ehrlichiosis, suggesting that E. canis infection should be considered a rather uncommon cause of arthritis in dogs.

Serum canine pancreatic lipase immunoreactivity in experimentally induced and naturally occurring canine monocytic ehrlichiosis (Ehrlichia canis).

Ehrlichia canis infection causes multisystemic disease in dogs (canine monocytic ehrlichiosis, CME) which is associated with variable morbidity and mortality. Atypical clinical manifestations, including gastrointestinal signs, may occasionally occur in CME and approximately 10-15% of dogs are presented with historical or clinical evidence of vomiting, diarrhea, and/or abdominal discomfort. The objective of this study was to investigate if there are any alterations in serum canine pancreatic lipase immunoreactivity (cPLI) in dogs with experimentally induced or naturally occurring monocytic ehrlichiosis. Serum samples from 10 Beagle dogs experimentally infected with E. canis and two healthy uninfected Beagles were serially examined; samples from 20 naturally infected dogs (10 with non-myelosuppressive [NME] and 10 with myelosuppressive [ME] ehrlichiosis) were also examined at a given point in time (cross-sectional sampling). None of the experimentally infected Beagles showed gastrointestinal signs or increased cPLI concentrations prior to or following the artificial infection. Three naturally infected dogs with NME and one with ME demonstrated serum cPLI concentrations in the diagnostic range for pancreatitis (>400 µg/L) without showing gastrointestinal signs. The results of the present study indicated that 4/20 (20%) of dogs naturally infected with E. canis demonstrated increased serum cPLI concentrations consistent with mild and clinically inapparent pancreatitis.

Efficacy of rifampicin in the treatment of experimental acute canine monocytic ehrlichiosis.

OBJECTIVES: To assess the efficacy of rifampicin in achieving clinical and haematological recovery and clearing infection in dogs with experimentally induced acute monocytic ehrlichiosis. METHODS: Five Ehrlichia canis-infected Beagle dogs were treated with rifampicin (10 mg/kg/24 h orally for 3 weeks), nine E. canis-infected dogs received no treatment (infected untreated dogs) and two dogs served as uninfected controls. Clinical score, platelet counts, immunofluorescent antibody titres and PCR detection of E. canis-specific DNA in blood, bone marrow and spleen aspirates were evaluated on post-inoculation days 21 (start of rifampicin), 42 (end of rifampicin) and 98 (end of the study). RESULTS: By day 21 post-inoculation, all infected dogs became clinically ill and thrombocytopenic, seroconverted and were PCR positive in at least one tissue. Clinical scores and antibody titres did not differ between the treated and infected untreated dogs throughout the study. The rifampicin-treated dogs experienced an earlier resolution of their thrombocytopenia (Kaplan-Meier survival plot, P=0.048), and the median platelet counts were significantly higher in the treated compared with the infected untreated dogs on post-inoculation days 42 (P=0.0233) and 98 (P=0.0195). At the end of the study, three treated and six untreated infected dogs remained PCR positive in one tissue each. CONCLUSIONS: The rifampicin treatment regimen applied in this study hastened haematological recovery, but was inconsistent in eliminating the acute E. canis infection.

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR.

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.

High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

Serum cardiac troponin I concentrations in naturally occurring myelosuppressive and non-myelosuppressive canine monocytic ehrlichiosis.

The aim of this study was to determine whether serum cardiac troponin I (cTnI) concentration measured on admission was associated with the clinical severity of canine monocytic ehrlichiosis and was predictive of clinical outcome (death or survival) in dogs naturally infected with Ehrlichia canis. Serum cTnI concentration was compared among 22 dogs with non-myelosuppressive ehrlichiosis (NME), 22 dogs with myelosuppressive ehrlichiosis (ME) and 10 healthy dogs. Unlike healthy dogs, 45.5% NME and 59.1% ME dogs had increased cTnI concentrations. There was no difference in the frequency of cTnI increase or mean cTnI concentrations between the NME and ME groups, whereas mean cTnI concentration was significantly lower in healthy dogs. No association was established between cTnI concentration on admission and clinical outcome.

The chlamydiales pangenome revisited: structural stability and functional coherence.

The entire publicly available set of 37 genome sequences from the bacterial order Chlamydiales has been subjected to comparative analysis in order to reveal the salient features of this pangenome and its evolutionary history. Over 2,000 protein families are detected across multiple species, with a distribution consistent to other studied pangenomes. Of these, there are 180 protein families with multiple members, 312 families with exactly 37 members corresponding to core genes, 428 families with peripheral genes with varying taxonomic distribution and finally 1,125 smaller families. The fact that, even for smaller genomes of Chlamydiales, core genes represent over a quarter of the average protein complement, signifies a certain degree of structural stability, given the wide range of phylogenetic relationships within the group. In addition, the propagation of a corpus of manually curated annotations within the discovered core families reveals key functional properties, reflecting a coherent repertoire of cellular capabilities for Chlamydiales. We further investigate over 2,000 genes without homologs in the pangenome and discover two new protein sequence domains. Our results, supported by the genome-based phylogeny for this group, are fully consistent with previous analyses and current knowledge, and point to future research directions towards a better understanding of the structural and functional properties of Chlamydiales.

Evolutionary relationships among Chlamydophila abortus variant strains inferred by rRNA secondary structure-based phylogeny.

The evolutionary relationships among known Chlamydophila abortus variant strains including the LLG and POS, previously identified as being highly distinct, were investigated based on rRNA secondary structure information. PCR-amplified overlapping fragments of the 16S, 16S-23S intergenic spacer (IS), and 23S domain I rRNAs were subjected to cloning and sequencing. Secondary structure analysis revealed the presence of transitional single nucleotide variations (SNVs), two of which occurred in loops, while seven in stem regions that did not result in compensatory substitutions. Notably, only two SNVs, in 16S and 23S, occurred within evolutionary variable regions. Maximum likelihood and Bayesian phylogeny reconstructions revealed that C. abortus strains could be regarded as representing two distinct lineages, one including the "classical" C. abortus strains and the other the "LLG/POS variant", with the type strain B577(T) possibly representing an intermediate of the two lineages. The two C. abortus lineages shared three unique (apomorphic) characters in the 23S domain I and 16S-23S IS, but interestingly lacked synapomorphies in the 16S rRNA. The two lineages could be distinguished on the basis of eight positions; four of these comprised residues that appeared to be signature or unique for the "classical" lineage, while three were unique for the "LLG/POS variant". The U277 (E. coli numbering) signature character, corresponding to a highly conserved residue of the 16S molecule, and the unique G681 residue, conserved in a functionally strategic region also of 16S, are the most pronounced attributes (autapomorphies) of the "classical" and the "LLG/POS variant" lineages, respectively. Both lineages were found to be descendants of a common ancestor with the Prk/Daruma C. psittaci variant. Compared with the "classical", the "LLG/POS variant" lineage has retained more ancestral features. The current rRNA secondary structure-based analysis and phylogenetic inference reveal new insights into how these two C. abortus lineages have differentiated during their evolution.

Genome sequence of the Chlamydophila abortus variant strain LLG.

Chlamydophila abortus is a common cause of ruminant abortion. Here we report the genome sequence of strain LLG, which differs genotypically and phenotypically from the wild-type strain S26/3. Genome sequencing revealed differences between LLG and S26/3 to occur in pseudogene content, in transmembrane head/inc family proteins, and in biotin biosynthesis genes.

Genetic variability of Chlamydophila abortus strains assessed by PCR-RFLP analysis of polymorphic membrane protein-encoding genes.

This study used PCR-RFLP to investigate the genetic variability of pmp-encoding genes from fifty-two Chlamydophila abortus (C. abortus) strains originating from abortion cases from various geographical regions and host species. Six primer pairs were used to PCR-amplify DNA fragments encoding eighteen pmps. PCR products were digested using four restriction endonucleases and Bayesian methodologies were used to compare RFLP profiles and assign strains to a RFLP genotype. Strains could be assigned to 2 genotypes in the region encoding pmp18D, 3 genotypes in the regions encoding pmp1A-pmp2B, pmp3E-pmp6H and pmp11G-pmp15G, 4 genotypes in the region encoding pmp7G-pmp10G and 5 genotypes in the region encoding pmp16G-pmp17G. In all regions, the majority of strains (88.4-96.1%) had the same genotype as the reference strain S26/3. No correlation could be made between genotype, host species or geographical origin except for the two variant Greek strains, LLG and POS, which formed a discrete genotype in all pmp-encoding regions except pmp18D. Relative rates of evolution calculated for each pmp-encoding gene locus suggest that differing selective pressures and functional constraints may exist on C. abortus polymorphic membrane proteins. These findings suggest that although intraspecies heterogeneity of pmp-encoding genes in C. abortus is low, the sequence heterogeneity should be an important consideration when using pmps as the basis for novel diagnostics or vaccine development.