Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts

Anàlisi polifàsica de soques de "Pseudomonas fluorescens" potencials agents de biocontrol de malalties de fruiters

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases. L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP. Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn. S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes. Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies. El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri. Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP. Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.

Fluorescence in situ hybridization with rDNA probes on chromosomes of two nucleolus organizer region phenotypes of a species of Eigenmannia (Pisces, Gymnotoidei, Sternopygidae)

Nucleolus organizer regions (NORs) were analysed in two related and geographically close populations of Eigenmannia sp.1 (Pisces, Gymnotoidei, Sternopygidae) using silver staining and fluorescence in situ hybridization (FISH). The two populations differed in their AS-NOR phenotypes, displaying fixed differences in the NOR-bearing chromosome pairs. FISH with rDNA probes showed that these differences were due to the location of rDNA cistrons. This finding, showing fixed NOR differences between two populations belonging to the same species in a connected river system, is highly significant in terms of evolutionary change, possibly indicating an initial step of genetic differentiation. This result also has important implications from the cytosystematic point of view, as NORs usually have a very constant karyotypic location in fish species and have been used as species-specific chromosome markers.

Cytogenetics of two species of Paratelmatobius (Anura: Leptodactylidae), with phylogenetic comments

In this paper we provide a cytogenetic analysis of Paratelmatobius cardosoi and Paratelmatobius poecilogaster. The karyotypes of both species showed a diploid number of 24 chromosomes and shared some similarity in the morphology of some pairs. On the other hand, pairs 4 and 6 widely differed between these complements. These karyotypes also differed in their NOR number and location. Size heteromorphism was seen in all NOR-bearing chromosomes of the two karyotypes. In addition, both karyotypes showed small centromeric C-bands and a conspicuous heterochromatic band in the short arm of chromosome 1, although with a different size in each species. The P. cardosoi complement also showed other strongly stained non-centromeric C-bands, with no counterparts in the P. cardosoi karyotype. Chromosome staining with fluorochromes revealed heterogeneity in the base composition of two of the non-centromeric C-bands of P. cardosoi. Comparison of the chromosomal morphology of these Paratelmatobius karyotypes with that of P. lutzii showed that the P. poecilogaster karyotype is more similar to that of P. lutzii than P. cardosoi. These cytogenetic results agree with the proposed species arrangements in the P. cardosoi and P. lutzii groups based on morphological and ecological data.

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cryl genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.

Localization of rDNA sites in holocentric chromosomes of three species of triatomines (Heteroptera, Triatominae).

Chromatin organization in the holocentric chromosomes of three triatomines species was cytologically studied by fluorescent in situ hybridization with a 45S rDNA probe of Drosophila melanogaster to localize ribosomal genes. In Triatoma tibiamaculata, metaphases I showed telomeric highlights in a single, larger bivalent. In T. protacta, hybridization was detected in one of the telomeres of an autosomal chromosome. In T. platensis, there were highlights in a single, smaller chromosome (X chromosome). The results obtained did not agree with the expected localization of rDNA genes in the sex chromosomes of triatomines, as demonstrated by silver impregnation, and suggest that the chromosome reorganization that occurred in this group during evolution may be a more important mechanism involved in rDNA distribution.

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed. © 2006 Cambridge University Press.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): New perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers. © 2011 Springer Science+Business Media B.V.

Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae)

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18. mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate. © 2011 Elsevier Inc.

Are NORs Always Located on Homeologous Chromosomes? A FISH Investigation with rDNA and Whole Chromosome Probes in Gymnotus Fishes (Gymnotiformes)

Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data. © 2013 Milhomem et al.

Fungos fitopatógenos associados às formigas cortadeiras (Formicidae: Tribo Attini)

The ants of genus Atta belong to the Attine tribe (order Hymenoptera, family Formicidae, subfamily Myrmicinae) and are commonly known as leaf-cutting ants for having the habit of cutting several vegetable species used as substrate for growing mutualistic fungus (Agaricales: Lepiotaceae). Recent studies showed that, in addition to that, other fungi may occur in the nests in a dorment state or participate in the functioning dynamic of this symbiosis. Researches related to surveys of fungus biodiversity in nests of different Atta species have found important phytopathogenic representatives. In Brazil, studies about integrated management of plagues, developed by Embrapa Meio Ambiente (Embrapa Environment), point out the need of higher investments in projects that involve the phytopathogenic transmission by insects in order to reduce costs to control them or minimize environmental impact. The purpose of this study was to broaden the knowledge about the ecology of these fungi, isolating and identifying species associated with Attine tribe ants, thus understanding the scope of pathogenic and phytopathogenic species spread by these ants. For that reason, gynes were collected from Atta laevigata and Atta capiguara anthills located at Unesp Botucatu (São Paulo, Brazil) campus. In order to isolate the fungus, the mineral oil floating technique was used. The identification of the isolated fungi was done based on microscopic and molecular characteristics using DNA ribosomal sequencing. The most highly abundant genera found so far were: Cladosporium, Exophiala, Penicillium, Acremonium, Phialophora and Teratosphaeria. Representatives of the genera Exophiala, Phialophora and Cladosporium may be human pathogens, whereas Teratosphaeria and Penicillium are related to diseases in Eucalyptus and citric fruits, respectively. The results show that these ants may host important fungal species besides the ones already... (Complete abstract click electronic access below)

A dysflagellar mutant of Leishmania (Viannia) braziliensis isolated from a cutaneous leishmaniasis patient

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features. Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo. Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis. Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.

COMPARISON OF PRIMERS FOR RAPD-PCR FROM ENVIRONMENTAL ISOLATES OF CRYPTOCOCCUS NEOFORMANS, CRYPTOCOCCUS ALBIDUS AND CRYPTOCOCCUS LAURENTII COMPLEX

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e. g., CAV2 for C. albidus) may not to be applied to some species.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.