Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome.

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Fatal coxiellosis in Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus).

Three Swainson's Blue Mountain Rainbow Lorikeets (Trichoglossus haematodus moluccanus), ranging from 6 to 8 months of age, presented with lethargy, emaciation, and progressive neurologic signs. The first one died 24 hours after the onset of clinical signs, and the other two were euthanized 10 to 14 days after the onset of progressive neurologic disease. Clinical signs in these lorikeets included head pressing, hemiparesis, seizures, obtunded mentation, weakness, and lethargy. Two of the lorikeets had hepatomegaly, and one had splenomegaly on gross examination. Histopathology revealed disseminated microgranulomas in the liver, spleen, and brain, and lymphohistocytic perivascular encephalitis and cephalic vasculitis. Electron microscopic examination of macrophages in brain lesions revealed spherical to rod-shaped prokaryotic organisms with a trilaminar cell wall. Molecular analysis revealed a novel species of Coxiella. This is believed to be the first report of a Coxiella sp. causing disease in a lorikeet.

Encephalitozoon cuniculi placentitis and abortion in a quarterhorse mare.

Encephalitozoon cuniculi is a microsporidial parasite, which has rarely been reported to cause placentitis in animals. A late-term aborted fetus and placenta from a Quarterhorse were presented to the Livestock Disease Diagnostic Center, University of Kentucky, for diagnostic examination. There was a necrotizing placentitis, with distension of many chorionic epithelial cells by intracytoplasmic vacuoles containing 1-2-microm-diameter, elongated, gram-positive organisms. The organisms were identified as E. cuniculi by electron microscopy and by polymerase chain reaction using primers to microsporidial ribosomal DNA. Joints of the fetus were swollen, with gross and microscopic lesions of synovitis; however, E. cuniculi DNA was not detected.

Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission.

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Polymerase chain reaction for definitive identification of Yersinia ruckeri.

Yersinia ruckeri causes enteric red mouth (ERM) disease in salmonids. Serologic identification of Y. ruckeri is hampered by cross-reactivity with other bacterial isolates of fish origin. Oligonucleotide primers incorporating Y. ruckeri unique sequences were designed to amplify a 409 bp fragment of Y. ruckeri 16S rDNA. The primers did not amplify other genetically related Yersinia or a wide variety of other aquatic or piscine bacteria. This assay provides a rapid, definitive identification of Y. ruckeri that is not subject to the variability inherent in serologic methods.

Monoclonal antibody binding to a surface-exposed epitope on Cowdria ruminantium that is conserved among eight strains.

Monoclonal antibodies (MAb) binding to Cowdria ruminantium elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with Ehrlichia canis antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development.

Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics.

Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginale and related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2 sequences to the promoter and polycistron.

Monoclonal antibody differentiation of Mycoplasma mycoides subsp. mycoides small-colony strains causing contagious bovine pleuropneumonia from less important large-colony strains.

Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine Mycoplasma mycoides subsp. mycoides small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for M. mycoides subsp. mycoides small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.

Streptococcus didelphis sp. nov., a streptococcus with marked catalase activity isolated from opossums (Didelphis virginiana) with suppurative dermatitis and liver fibrosis.

beta-Haemolytic, catalase-positive, Gram-positive cocci that formed chains in broth media but did not react with Lancefield group antisera were isolated from skin lesions, spleen, liver and lungs of nine opossums, including eight from a research colony and one from a wildlife rehabilitation organization. The isolates had vigorous catalase activity that was retained on initial passage on non-blood-containing media, but this activity was lost in subsequent passages. The use of standard phenotypic tests did not lead to satisfactory identification of these organisms beyond the genus level, even if the aberrant catalase reaction was not considered. The 16S rRNA gene sequence of the isolates was most similar (96%) to Streptococcus dysgalactiae, but distinct from that species as 16S rRNA gene similarity of different strains of S. dysgalactiae was > 99%. Characterization of biochemical reactions and cell-wall fatty acid profiles also revealed significant differences between the opossum isolates and all other known Streptococcus spp., thus it is proposed as a new species with the name Streptococcus didelphis, sp. nov. The type strain is ATCC 700828T.

Strain diversity in major surface protein 2 expression during tick transmission of Anaplasma marginale.

Specific major surface protein 2 (MSP2) variants are expressed by Anaplasma marginale within the tick salivary gland and, following transmission, are expressed during acute rickettsemia. In previous work, we have shown that a restricted pattern of MSP2 variants is expressed in the salivary glands of Dermacentor andersoni ticks infected with the South Idaho strain of A. marginale. Now we demonstrate that the identical restriction does not apply to two other strains of A. marginale, and that different variants are also expressed when the same strain is transmitted by different Dermacentor spp. This indicates that antigenic diversity among strains is maintained in tick transmission and may be a significant constraint to MSP2 vaccine development.

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

Molecular basis for vaccine development against the ehrlichial pathogen Anaplasma marginale.

Anaplasma marginale is a tick-transmitted ehrlichial pathogen causing severe morbidity and mortality in livestock on six continents. Development of safe effective vaccines would be greatly facilitated by identification of the protective immune mechanisms and by understanding how the pathogen evades immune effectors to establish persistent infection. In this article, Guy Palmer and colleagues review recent progress in identifying how defined epitopes induce protective immunity and the role of antigenic variation in these epitopes as a mechanism of persistence.

Analysis of the 16S rRNA gene of micro-organism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

The structural gene encoding the 16S rRNA of the new obligate intracellular organism presently designated WSU 86-1044T was sequenced and analysed to establish its phylogenetic relationships. The 16S rDNA sequence was most closely related to those of chlamydial species, having 84.7-85.3% sequence similarity, while it had 72.4-73.2% similarity with rickettsia-like organisms. When the sequences of the four species of chlamydiae (Chlamydophila psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila pecorum) were compared, they had > 93% sequence similarity indicating that WSU 86-1044T was not close enough to be in the same family as current Chlamydiaceae members. However, based on the 84.7-85.3% 16S rDNA sequence similarity of WSU 86-1044T and other previously described characteristics, WSU 86-1044T belongs to a novel family within the order Chlamydiales; hence, the proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

Restriction of major surface protein 2 (MSP2) variants during tick transmission of the ehrlichia Anaplasma marginale.

Anaplasma marginale is an ehrlichial pathogen of cattle that establishes lifelong persistent infection. Persistence is characterized by rickettsemic cycles in which new A. marginale variant types, defined by the sequence of the expressed msp2 transcripts, emerge. The polymorphic msp2 transcripts encode structurally distinct MSP2 proteins and result in an antigenically diverse and continually changing A. marginale population within the blood. In this manuscript, we used sequence analysis of msp2 transcripts to show that a restricted repertoire of variant types, designated SGV1 and SGV2, is expressed within the tick salivary gland. The same SGV1 and SGV2 variant types were expressed in ticks regardless of the variant types expressed in the blood of infected cattle at the time of acquisition feeding by the ticks. Importantly, subsequent tick transmission to susceptible cattle resulted in acute rickettsemia composed of organisms expressing only the same SGV1 and SGV2 variant types. This indicates that the msp2 expressed by organisms within the tick salivary gland predicts the variant type responsible for acute rickettsemia and disease. This restriction of transmitted A. marginale variant types, in contrast to the marked diversity within persistently infected cattle, supports development of MSP2 vaccines to prevent acute rickettsemia in tick-transmitted infections.

A DNA vaccine protects mice against the rickettsial agent Cowdria ruminantium.

A DNA vaccine (VCL1010/MAP1) containing the major antigenic protein 1 (MAP1) gene of Cowdria ruminantium, driven by the human cytomegalovirus (HCMV) enhancer-promoter, was injected intramuscularly into 8-10 week-old female DBA/2 mice after treating them with 50 microliters/muscle of 0.5% bupivacaine three days previously. Up to 75% of the immunized mice seroconverted and reacted with C. ruminantium antigen blots. Splenocytes from immunized mice, but not from control mice, proliferated in response to the recombinant MAP1 and to C. ruminantium antigens in in vitro lymphocyte proliferation tests. These proliferating cells secreted IFN-gamma and IL-2 at concentrations ranging from 610 pg/ml to 1290 pg/ml and from 152 pg/ml to 310 pg/ml, respectively. Only up to 45 pg/ml and 42 pg/ml of IFN-gamma and IL-2, respectively, were detected in supernatants of splenocytes from control mice. In experiments testing different VCL1010/MAP1 DNA vaccine dose regimens (25-100 micrograms/dose, two or four immunizations), survival rates of 23% to 88% (35/92 survivors/total in all VCL1010/MAP1 immunized groups) were observed on challenge with a lethal dose of cell culture-derived C. ruminantium organisms. In contrast, survival rates of 0% to 3% (1/144 survivors/total in all control groups) were recorded for control mice. This study demonstrates that MAP1 is a protective antigen and validates the concept of DNA vaccines against heartwater.

Monoclonal antibody E8-18 identifies an integral membrane surface protein unique to Mycoplasma capricolum subsp. capripneumoniae.

Monoclonal antibody (MAb) E8-18 reacted with four isolates of Mycoplasma capricolum subsp. capripneumoniae in Western blots identifying an epitope on a 24 kDa antigen (p24). MAb E8-18 did not react with 11 isolates belonging to four other Mycoplasma species or subspecies closely related to M. capricolum subsp. capripneumoniae. A combination of trypsin treatment of intact organisms and detergent-phase partitioning revealed p24 to be an integral M. capricolum subsp. capripneumoniae surface membrane protein.

Monoclonal antibodies to surface-exposes proteins of Mycoplasma mycoides subsp. mycoides (small-colony strain), which causes contagious bovine pleuropneumonia.

Outbreaks of bovine pleuropneumonia caused by small-colony strains of Mycoplasma mycoides subsp. mycoides occur in Africa, and vaccination is used for control. Since protein subunits are needed to improve multivalent vaccines, monoclonal antibodies (MAbs) were made to facilitate protein identification and isolation. Eleven immunoglobulin M MAbs derived from mouse spleen donors immunized with disrupted whole organisms bound periodate-sensitive epitopes on externally exposed polysaccharide. Seven of these MAbs caused in vitro growth inhibition of M. mycoides subsp. mycoides; however, reaction with carbohydrate epitopes prevented their use in identifying proteins. Ten additional MAbs from mouse spleen donors immunized with Triton X-114-phase integral membrane proteins reacted with periodate-insensitive, proteinase K-sensitive epitopes. These MAbs were classified into three groups based on immunoblots of Triton X-114-phase proteins. One group reacted with 96-, 16-, and 15-kDa proteins. Another group reacted with 26-, 21-, and 16-kDa proteins, while a third group reacted only with 26- and 21-kDa proteins. One MAb from each group reacted with trypsinsensitive epitopes on live organisms, yet none caused in vitro growth inhibition. Representative MAbs reacted with all small-colony strains in immunoblots and did not react with large colony strains. However, these MAbs were not specific for small-colony strains, as proteins from two other M. mycoides cluster organisms were identified. Nevertheless, MAbs to surface-exposed epitopes on integral membrane proteins will be useful for isolation of these proteins for immunization, since one or more might induce growth-inhibiting antibodies or other protective responses.

Effect of anti-thymocyte serum on acquisition of resistance to infestation by Rhipicephalus appendiculatus larvae in rabbits.

Administration of specific goat anti-thymocyte serum (ATS) to rabbits, prior to a primary infestation by Rhipicephalus appendiculatus larvae, blocked the acquisition of resistance significantly only in the third infestation. The larvae which fed on these rabbits had higher engorgement masses than did those feeding on untreated control rabbits. Also, a higher percentage (92%) of larval ticks fed on these animals than on the controls (88%). ATS also induced a leucopenia due to a lymphopenia in the treated rabbits. It was concluded that a T-cell-dependent component might be involved in acquired resistance to infestation by R. appendiculatus.