Extreme Cranial Ontogeny in the Upper Cretaceous Dinosaur Pachycephalosaurus

John R Horner
Museum of the Rockies
Montana State University
BozemanMontanaUnited States of America

Mark B Goodwin
Museum of Paleontology
University of California
BerkeleyCalifornia

Extreme Cranial Ontogeny in the Upper Cretaceous Dinosaur Pachycephalosaurus
United States of America
Background: Extended neoteny and late stage allometric growth increase morphological disparity between growth stages in at least some dinosaurs. Coupled with relatively low dinosaur density in the Upper Cretaceous of North America, ontogenetic transformational representatives are often difficult to distinguish. For example, many hadrosaurids previously reported to represent relatively small lambeosaurine species were demonstrated to be juveniles of the larger taxa. Marginocephalians (pachycephalosaurids + ceratopsids) undergo comparable and extreme cranial morphological change during ontogeny.Methodology/Principal Findings: Cranial histology, morphology and computer tomography reveal patterns of internal skull development that show the purported diagnostic characters for the pachycephalosaurids Dracorex hogwartsia and Stygimoloch spinifer are ontogenetically derived features. Coronal histological sections of the frontoparietal dome of an adult Pachycephalosaurus wyomingensis reveal a dense structure composed of metaplastic bone with a variety of extremely fibrous and acellular tissue. Coronal histological sections and computer tomography of a skull and frontoparietal dome of Stygimoloch spinifer reveal an open intrafrontal suture indicative of a subadult stage of development. These dinosaurs employed metaplasia to rapidly grow and change the size and shape of their horns, cranial ornaments and frontoparietal domes, resulting in extreme cranial alterations during late stages of growth. We propose that Dracorex hogwartsia, Stygimoloch spinifer and Pachycephalosaurus wyomingensis are the same taxon and represent an ontogenetic series united by shared morphology and increasing skull length.

Introduction

Pachycephalosaurids are a group of ornithischian dinosaurs united by the presence of cranial ornamentation and an enlarged frontoparietal dome, a unique morphology among vertebrates. The history and description of Pachycephalosaurus involved many prominent dinosaur paleontologists of the early 20 th century. A review of their early observations and taxonomy, together with the systematic paleontology of Pachycephalosaurus proposed in this study, is provided in Supporting Information [Text S1].

A great many papers have been written about pachycephalosaurid crania, in particular concerning the variability of the frontoparietal domes [1,2 and references cited therein], but there are few studies that have attempted to understand the development of these unusual cranial structures. Interestingly, even though ontogeny was discussed in a few of these evaluations [3][4][5][6][7][8], it was not considered an important variant beyond the relative inflation of the dome, apparently because of the relatively similar sizes of the different individuals from time-equivalent strata. The presumption that individuals possessing juvenile characteristics are expected to be much smaller than adults is not corroborated in hadrosaurs [9] and ceratopsian dinosaurs [10][11][12] with a sufficient sample size. Prior evaluations of cranial variation in pachycephalosaurids did not consider earlier studies that demonstrated several dinosaur taxa showed allometric growth of the skull and a significant expression of horns, domes and bony ornaments when the skulls reached approximately 50% size. Now, with a greater number of pachycephalosaurid skulls from the Upper Cretaceous of the Western Interior of North America available for study, and the use of comparative cranial morphology, histology and computer tomography, multiple lines of evidence support our alternative hypothesis that Dracorex hogwartsia and Stygimoloch spinifer represent earlier growth stages in a single taxon, Pachycephalosaurus wyomingensis.

Relative, or proportional growth (allometry of Huxley [13]), refers to shape change with regard to an increase in size. Brown and Schlaikjer [10] first reported this relative growth in dinosaur crania in the Cretaceous Mongolian ceratopsid Protoceratops. Later studies by Rozhdesvensky [14] found the phenomenon not only in Protoceratops, but also in the Mongolian hadrosaurid Saurolophus and theropod Tarbosaurus. Allometric growth was apparent in these taxa, in part, because of the large number of specimens representing a range of ontogenetic transitions collected from the Mongolian strata. Dinosaurs of the Upper Cretaceous of Mongolia are tremendously abundant and this region is recognized around the world as one of the planet's most fertile collecting grounds. Due to relatively lower specimen density in the Western Interior of North America, ontogenetic transformational representatives were difficult to distinguish until Dodson [9] performed statistical studies on a group of ''closely related'' lambeosaurine hadrosaurs from Upper Cretaceous strata in Alberta, Canada, in 1975. Dodson's study revealed that many hadrosaurids previously reported to represent relatively small lambeosaurine species were instead juveniles of the larger taxa. These lambeosaurine taxa experienced cranial allometric growth and retained juvenile characters (neoteny) until the skulls reached approximately 50% adult size. Dodson [9] compared the retarded development of the characteristic hollow narial crest in lambeosaurines with the expansion of the casque in the avian taxon Casuarius. Casque development begins after the cassowary skull reaches 65% to 80% adult size. Dodson showed that extended neoteny and late stage allometry increased the morphological disparity between particular growth ''stages'' within the lambeosaurine taxa. In 1976, Dodson [11] published a related study on Protoceratops, and provided statistical data to quantify the earlier qualitative observations on allometric growth in these dinosaur skulls [10,14].

Morphogenetic and osteohistogenetic evaluations of dinosaur genera from the Upper Cretaceous Hell Creek Formation of Montana, North and South Dakota, and equivalent age sediments in adjacent states and Canadian Provinces, are providing evidence that extreme modification of dinosaur skulls occurs when these skulls approach $50% adult size in many dinosaur groups, particularly the Marginocephalia. Marginocephalian dinosaur growth and histology of their cranial ornamentations follow the predicted trend of osteohistogenesis. A Triceratops ontogenetic series described by Horner and Goodwin [15,16] revealed major ontogenetic modifications to cranial ornamentations on various parts of the skulls. The epiparietals and episquamosals bordering the edge of the parietalsquamosal frill are initially diminutive before expanding to larger, taller equilateral triangular bones, and later in ontogeny, flatten dorsoventrally and elongate as they merge with the edge of the frill. The smallest epiparietals, episquamosals and postorbital horns reveal the youngest histological tissues. Postorbital horns that grew straight at first, arc strikingly backward in younger individuals then recurve forward in later stages of ontogeny. Forward horn orientation and expression of the largest epiparietals and episquamosals occur in crania that are about 65% the length of the largest known specimens. We confirm a similar pattern of development and osteohistogenesis in the cranial morphology of the squamosal horns, nodes and frontoparietal dome in the skulls of the pachycephalosaurids Dracorex hogwartsia, Stygimoloch spinifer and Pachycephalosaurus wyomingensis.

Results and Discussion

In the following section, we begin with Pachycephalosaurus and demonstrate that the nominal pachycephalosaurid genera, Dracorex and Stygimoloch, represent the same taxon at an earlier ontogenetic stage. We use comparative cranial histology and morphology to confirm how these ontogenetic transformations developed in a single taxon, Pachycephalosaurus (Table 1). These growth stages reflect a growth continuum rather than specific developmental steps defined by ''known'' terminal morphologies. This is important because currently we do not know the ultimate size or maximum age of any dinosaur species and an adult growth stage assignment purports a potentially false terminal morphological state. The frontoparietal and lateral cranial elements are highly inflated in the largest, and presumably oldest domed Pachycephalosaurus and follow the morphological sequence proposed for Stegoceras [3,17]. The frontoparietal dome was not preserved with the isolated left squamosal and holotype of S. spinifer, UCMP 119433 ( Figure 1A), but a dome is present in more complete pachycephalosaurid skulls with identical squamosals and squamosal horns referred to S. spinifer such as UCMP 131163 ( Figure 1B Figure 4) that is evidence of cranial vault expansion during postnatal craniofacial growth [18]. The intrafrontal suture functions as an intramembranous bone growth site and remains unossified as new bone is formed [18] in this subadult ontogenetic stage. The absence of a visible intrafrontal suture on the dorsal skulls of ''Stygimoloch'' previously obscured their relative age.

In Figure 3, morphological landmarks are colored and evident on the skulls of Pachycephalosaurus, Dracorex and Stygimoloch. Inflation of the frontoparietal dome and corresponding closure of the temporal fenestrae are two major morphological changes that occur between Dracorex and Pachycephalosaurus with concurrent lengthening of the skull that ontogenetically separates the three pachycephalosaurids older than ''Dracorex'' shown in Figure 3A-F (see Table 1). A composite image of the holotype of Dracorex    Figure 5. The combined cranial morphology of this subadult ontogenetic stage was verified by the authors and Sullivan [8:359] in two skulls from the Hell Creek Formation, Garfield County, Montana, that are privately held and currently unavailable for study. UCMP 556078 ( Figure 3C,D) is a slightly more advanced subadult stage of ontogeny, compared to the composite skull in Figure 5, with an inflated frontoparietal and lateral elements that are enlarged but not yet incorporated into the dome as they are in Pachycephalosaurus. We predict this ontogenetic trend, if projected backward toward an even younger end member than ''Dracorex'', would result in a flat-headed morph ,43 cm long ( = midline length of ''Dracorex'') with a slightly thickened (or incipiently domed) frontoparietal region, open supratemporal fenestrae and dorsal skull covered with emerging nodal ornamentation with clusters of relatively smaller diameter pyramidal nodes on the squamosals. A slightly less robust, narrower skull compared to the holotype of ''Dracorex'', with these precise morphological characteristics, is figured in the literature [19:6] and discussed by Sullivan [8]; however it is privately held.

A series of prominent pyramidal nodes on the anterodorsal surface of the nasals in the holotype of Dracorex hogwartsia ( Figure 3G,H) match the nasal ornaments preserved in the AMNH 1696 skull of Pachycephalosaurus wyomingensis ( Figure 3A,B) [8] and a slightly younger P. wyomingensis skull ( Figure 3C,D). We concur with Bakker et al. [20:331] that the ''long snout, two halfrings of pyramidal spikes on the snout…'' of ''Dracorex'' are nearly identical to the arrangement observed in Pachycephalosaurus and ''Stygimoloch''. The distinctive cluster of squamosal horns undergoes allometric growth before they are reduced in subadults. The slightly curved squamosal horns and nodes increase in length ontogenetically from ''D.'' hogwartsia ( Figure 3G,H) to ''S.'' spinifer ( Figure 3E,F) before the horns erode and nodes are modified into their blunt but robust pyramidal shape in the ontogenetically most advanced skulls of P. wyomingensis ( Figure 3A,B). The nodes form asymmetrical clusters on the dorsoposterior surface of the squamosals in the adult Pachycephalosaurus ( Figure 6, Figure 7). Just as the number, arrangement and shape of the epiparietals and episquamosals vary between individual Triceratops [16], the variability in the arrangement, dimensions and symmetry of the nodal ornamentation on the posterior skull margin in Pachycephalosaurus is neither unexpected nor unique within the Marginocephalia [8,21]. While a strict 1:1 correspondence between the squamosal ornamentation in ''Dracorex'' and Pachycephalosaurus is not exact, it is clearer between ''Dracorex'' and ''Stygimoloch'' before the frontoparietal undergoes extreme ontogenetic modification, particularly in the posterior-most region of the skull with the expansion of the cranial vault. Individual variation within this ontogenetic continuum plays a major role in the morphology of this highly modified region of the skull. Alternatively, these ''morphs'' could represent different taxa, but evidence from comparative cranial histology, computer tomography, morphology, and similar patterns of growth observed in Stegoceras [17] and Triceratops [15,16] does not support this interpretation.  Giffin's study [7] of seven pachycephalosaurids assesses geometric growth and relative frontoparietal dome shape using two landmarks restricted to the frontoparietal dome in a geographically diverse sample too small for statistical testing.

Cranial Histology

Bone histology is a valuable tool for assessing various growth parameters including relative growth rate and general bone ontogeny. Radial vascularization, for example, ''…is functionally associated with very fast deposition of relatively modest amounts of new compact bone'' [22:512]. Additionally, woven bone with minimal osteonal deposition is indicative of early osteogenesis, whereas dense Haversian or reconstructed tissues are indicative of later osteogenesis [17]. We utilize these general histological observations to ontogenetically order the various pachycephalosaurid cranial characteristics described in this study (Table 1,  Table 2).

Histological examination of the cranial ornamentations of dinosaurs reveals complex morphologies composed primarily of tissue formed during metaplasia. Metaplasia is the process in which dense fibrous connective tissues are transformed directly into bone without the intervention of a periosteum or the presence of osteoblasts [23]. Metaplastic bone, particularly as seen in dinosaur crania, has a wide variety of forms, from highly porous to extremely dense. These tissues can appear very similar to bone deposited by a periosteum in that they contain vascular canals, osteocyte-like lacunae and can be reworked by Haversian-like innervations. Although similar in appearance, the structures that look like osteocytes lack canaliculi, and instead possess stubby lateral processes that do not connect to adjacent cells [24]. These structures are herein referred to as fibrocytes and apparently represent trapped fibroblasts. Metaplastic bone is common in  reptiles [25], but generally described in association with osteoderms [25][26][27][28][29] and ossified tendons [30]. The metaplastic tissues in the skulls of pachycephalosaurids vary greatly in gross appearance depending on the cranial structure in which they form. As observed for other bone tissues, these metaplastic tissues undergo a consistent, predictable development in that the younger tissues are much more vascularized compared to increasingly mature tissues [17].

A coronal histological section of a pachycephalosaurid skull previously referred to ''Stygimoloch'' (UCMP 128383) shows an open intrafrontal suture in coronal view ( Figure 8A). The highly vascularized nature of the frontoparietal dome showing vascular spaces oriented primarily in a radial pattern ( Figure 8B) suggests that the dome was actively growing at a rapid pace [22:512]. Abundant fibers and fibrocytes located at the dorsal surface of the dome indicate an area of apparent inflation and ossification ( Figure 8C). A fully formed dome referable to Pachycephalosaurus ( Figure 8D) is much denser with comparatively less vascularization suggesting that growth has subsided [17]. Extending over the entire outer-most margin of the dome interior are the ends of fibers that apparently connected to a fibrous epidermal covering the dome ( Figure 8E). The presence of these exposed fibers suggests the dome was still inflating as there is no evidence of an erosional surface. A node on the postorbital bar ( Figure 8F), by contrast, possesses a surface that is erosional ( Figure 8G), bluntshaped and lacks any evidence of attachment or growth. An overlying matrix preserves this degraded surface. The most interesting and controversial aspect of our hypothesis, that ''Dracorex'' and ''Stygimoloch'' represent younger ontogenetic stages of Pachycephalosaurus, relates to the squamosal horns. They are medium to large pointed structures in ''Dracorex'' [20] and ''Stygimoloch'' (Figure 1, Figure 2), but are relatively shorter, blunt to lightly pointed and robust in Pachycephalosaurus (Figure 6,  Figure 7). Three squamosal horns were sectioned to establish this sequence, including the holotype of ''Stygimoloch'' (UCMP 119433). If ''Dracorex'' and ''Stygimoloch'' represent ontogenetic stages of Pachycephalosaurus, then it must be shown that these horns grow and elongate in ''Dracorex'' and ''Stygimoloch'' before the onset of erosion when they are reduced in size and modified in shape. We do not know at what growth stage maximum squamosal horn length is achieved, but it appears to occur between the holotype of ''Dracorex'' and some stage of ''Stygimoloch''.

The smallest horn (UCMP 128383) was found associated with the ''Stygimoloch'' dome in Figure 8A, revealing evidence of rapid dome growth. This horn also reveals highly vascularized tissue ( Figure 9A) indicative of rapid expansion in size and an extremely irregular exterior surface ( Figure 9B) that is very similar to bone that contacts a periosteum. The absence of osteocytes and abundance of fibers indicates the bone is expanding through metaplasia rather than periosteal ossification. A cross section through the largest central horn (#1 sensu Galton and Sues [31]) from the holotype of ''Stygimoloch'' ( Figure 1A) reveals metaplastic tissue with an embayed exterior surface ( Figure 9C) that is being eroded ( Figure 9D). There is no evidence that metaplasia was occurring at the time of the animal's death; however, there is an indication histologically that the horn underwent an earlier period of erosion followed by a phase of metaplasia, and subsequently a final period of erosion that was occurring when the animal died. Evidence for this sequence is the erosional unconformity indicated by the arrows in Figure 9C.

Another horn (MOR 560; found isolated) cut both longitudinally ( Figure 10A) and transversely ( Figure 10B) is also in an erosion mode as evidenced by a surface that is degrading with no signs of metaplasia ( Figure 10C). The primary metaplastic veneer is very thin, and most of the bone has been reconstructed by secondary innervations. Sharpey's fibers near the exterior surfaces and indented vessel channels on the exterior surfaces of each of the sectioned horns suggest that they were keratin covered regardless of whether they were undergoing metaplasia or erosion.

Conclusions

We propose that Dracorex hogwartsia and Stygimoloch spinifer are growth stages of Pachycephalosaurus wyomingensis and represent an ontogenetic series of P. wyomingensis united by shared morphology and increasing skull length. The youngest and most complete skull of Pachycephalosaurus yet known belongs to the growth stage best illustrated by ''Dracorex''. This synonymy significantly reduces the number of Upper Cretaceous pachycephalosaurid taxa.

Timing of dome inflation is probably highly variable based on the cranial patterns preserved in Stegoceras [4,32] and Triceratops [15,16]. Like some hadrosaurids and ceratopsids, these pachycephalosaurids retain their juvenile cranial characters until they are at least 50% grown. This ontogenetic pattern contributed to the naming of some new pachycephalosaurid taxa based on diagnostic characters (large supratemporal fenestrae, squamosal horns, nodal cranial ornamentation, inflated frontoparietal dome) that we demonstrate in this study are, in fact, ontogenetic features that undergo extreme modification as the skull length increases, the cranial vault enlarges and the frontoparietal dome expands. The squamosal horns and nodes of Pachycephalosaurus underwent a similar transformation to the epiparietal and episquamosal elements of Triceratops [16: figure 2] that (1) grew from diminutive to large triangular-shaped ornaments; (2) eroded as they reduced dorsoventrally and (3) flattened and lengthened as they merged onto the edge of the parietal-squamosal frill. Pachycephalosaurid horns originated ontogenetically as nodelike structures, some of which expanded into horns and bony spikes and then became modified as blunt, nodal structures in late stage ontogeny. The various shapes of the frontoparietal dome, from the characteristic tall broad dome of the Pachycephalosaurus wyomingensis (AMNH 1696) to the relatively flattened morphology of Pachycephalosaurus ''reinheimeri'' (DMNS 469) also reflect a comparable morphological transformation that occurs ontogenetically.

Marginocephalian dinosaurs employ metaplasia to grow their horns, cranial ornaments, domes and shields as they rapidly remodel their skulls. Any hypothesis testing of head butting in pachycephalosaurids, combat in ceratopsids or similar agonistic encounters must take into account the fact that the skulls of these dinosaurs are composed largely of metaplastic tissue.

Materials and Methods

Twenty-one pachycephalosaurid skulls and cranial elements examined in this study are listed in Table 2. Four casts were included to confirm external morphologies and supplement fossil specimens used for histological analysis. Cut blocks of fossils were embedded in Silmar-40 polyester resin prior to histological sectioning and mounted to glass slides with epoxy resin. Archival casts were made of the specimens before cutting and are catalogued in the collections of the MOR and UCMP. The thin sections were ground on a Buehler Ecomet grinder and sections were studied by light microscopy under normal and polarized light. Histological sections from this study are catalogued into the slide libraries of the MOR and UCMP. Some of the histological slides were prepared for a previous study by the authors [17].

Supporting Information

Figure 1 .
1The distinctive squamosal ornamentation and relatively high, narrow frontoparietal dome of Stygimoloch spinifer. The holotype left squamosal (UCMP 119433) in (A) and a right squamosal (UCMP 131163) in (B) in posterior view. UCMP 131163 was found associated with the relatively high, narrow frontoparietal dome in right lateral view in (C). The frontoparietal suture is highlighted in white. The intrafrontal suture is not visible on the dorsal surface. Numbering sequence (1-3) of horns and nodes in (A) and (B) from Galton and Sues [31]. Scale bars are 5 cm. doi:10.1371/journal.pone.0007626.g001

Figure 2 .
2The skull of Stygimoloch spinifer, MPM 8111, in right lateral view. This skull revealed the low angle orientation of horn #1 and surrounding nodes on the elongated squamosal shelf. Scale bar is 5 cm. doi:10.1371/journal.pone.0007626.g002

Figure 3 .
3Cranial ontogenetic sequence of Pachycephalosaurus wyomingensis with morphological landmarks highlighted in color. The ontogenetically oldest adult, AMNH 1696, in (A) dorsal and (B) right lateral views. A younger adult, UCMP 556078 (cast) with inflation of the frontoparietal dome+lateral cranial elements and mature nasal and squamosal nodal ornamentation in (C) dorsal and (D) right lateral views. MPM 8111, a partial skull of ''Stygimoloch'' in (E) dorsal and (F) left lateral views (reversed) illustrates the high narrow frontoparietal dome, squamosal nodes and horns characteristic of the subadult growth stage. Landmarks on the dorsal skull of MPM 8111 in orange (anterior) and red (posterior) constrain the position of the dome. The youngest growth stage in this cranial ontogenetic series is ''Dracorex'', TCNI 2004.17.1 (cast) in (G) dorsal and (H) right lateral views. The position of the squamosal horns and nasal nodes are consistent in these four pachycephalosaurid skulls, which increase in overall length and size from youngest (G,H) to oldest (A,B). Scale bar is 5 cm. doi:10.1371/journal.pone.0007626.g003 (TCMI 2004.17.1) and the most complete skull known of Stygimoloch (MPM 8111) catalogued in a repository shows the frontoparietal dome of MPM 8111 aligned with the skull of Dracorex (Figure 5). The composite morphology illustrates the considerable missing anterior portion of MPM 8111 that if not taken into consideration, gives the incorrect impression that this domed ''Stygimoloch'' is significantly shorter in length than ''Dracorex''. The preserved morphology of MPM 8111 is limited to the emerging frontoparietal dome and elongated horned squamosals. Landmarks confirm the position of the incipient dome on the ''Dracorex-Stygimoloch'' morph in

Figure 4 .
4A coronal CT scan through the dome of ''Stygimoloch'' (MPM 8111). The intrafrontal suture (black arrow) is open internally supporting the subadult status of this pachycephalosaurid. The braincase (bc) and foramen magnum (fm) are clearly visible. doi:10.1371/journal.pone.0007626.g004

Figure 5 .
5A composite image of ''Stygimoloch'' (MPM 8111) and ''Dracorex'' (TCNI 2004.17.1; cast) illustrates the subadult cranial ontogenetic stage morphology. The composite is constrained by landmarks on the skulls showing the position of the inflated frontoparietal dome of ''Stygimoloch'' on the posterior dorsal surface of ''Dracorex'' in (A) dorsal and (B) right lateral views. Scale bars are 5 cm. doi:10.1371/journal.pone.0007626.g005

Cranial ornamentation, presence or absence of morphological features and incorporation of lateral elements into the dome cannot be confirmed in this sample. Nonetheless, four specimens co-occur in the Hell Creek Formation, Carter Co., Montana. Of these, AMNH 1696 (adult Pachycephalosaurus), USNM 264304 (adult Pachycephalosaurus), and CCM 87-1 (''Stygimoloch'') support our ontogenetic hypothesis for Pachycephalosaurus. The fourth skull is Stegoceras edmontonensis ( = Sphaerotholus buchholtzae Williamson and Carr 2002; Prenocephale edmontonensis Sullivan 2003) and confirms these genera are sympatric in this assemblage.

Figure 6 .
6The holotype adult skull of Pachycephalosaurus ''reinheimeri'' (DMNS 469). (A) Asymmetrical clusters of massive, slightly pointed to rounded nodal ornaments on the dorsal surface of the squamosals dominate the posterior skull. (B) The squamosal nodes in posterior view. Scale bars are 5 cm. doi:10.1371/journal.pone.0007626.g006

Figure 7 .
7An adult Pachycephalosaurus skull (VRD 13) with ontogenetically advanced squamosal ornamentation. The adult cranial morphology and clusters of squamosal nodes in (A) and a close-up of the right squamosal ornamentation in (B) in right lateral views. doi:10.1371/journal.pone.0007626.g007

Figure 8 .
8Craniohistological coronal sections of ''Stygimoloch'' (A-C) and Pachycephalosaurus (D-F) skulls. (A) UCMP 128383.PSF-3, a coronal section through the frontoparietal dome clearly shows the open intrafrontal suture (white arrow) in this subadult ''Stygimoloch''. Scale bar is 2 cm. (B) UCMP 128383.PSF-3, a view of the middle region of the frontoparietal dome with highly vascularized tissue and vascular spaces oriented in a radial pattern. Scale bar is 1 mm. (C) UCMP 128383.PSF-3, the dorsal-most region of the frontoparietal dome reveals abundant fibers and fibrocytes present when the frontoparietal dome inflates and ossifies. Scale bar is 100 mm. (D) Coronal section of ''Pachycephalosaurus'', VRD 13. The denser and less vascularized tissue throughout this late growth stage suggests subsidence of frontoparietal dome growth. Black arrow points to postorbital nodal ornament sectioned in (F) and (G). Scale bar is 2 cm. (E) VRD 13, the ends of fibers (white arrows) extend into the outermost margin of the interior of the dome and likely connected to a fibrous epidermal covering. Scale bar is 100 mm. (F) VRD 13, a postorbital nodal ornament in transverse section lacks any evidence of an epidermal attachment or growth dorsally. Scale bar is 1 mm. (G) VRD 13, the erosional surface (white arrows) points to continued modification in size and shape of this postorbital nodal ornament. No evidence of an epidermal attachment or growth dorsally in the degraded surface preserved in the overlying tissue. Scale bar is 100 mm. doi:10.1371/journal.pone.0007626.g008

Figure 9 .
9Histological sections of ''Stygimoloch'' squamosal horns. (A) UCMP 128383.CrSp.1-2, transverse section of a highly-vascularized, expanding ''Stygimoloch'' squamosal horn found associated with a frontoparietal dome (see Fig. 3A) and partial skeleton. Scale bar is 2 cm. (B) UCMP 128383.CrSp.1-2, the red arrows point to the interior depositional surface and expansion of this squamosal horn. This metaplastic tissue closely resembles bone that contacts a periosteum, but the absence of osteocytes and abundant fibers indicates the horn is expanding through metaplasia rather than periosteal ossification. Scale bar is 100 mm. (C,D) UCMP 119433.SqSp.1-4, transverse sections reveal metaplastic tissue in the largest squamosal horn from the holotype of ''Stygimoloch'' (UCMP 119433). In (C), the interior erosional line is interrupted by a depositional surface along the red arrows. Scale bar is 2 cm. In (D), the erosional surface (red arrow) indicates the horns are getting smaller. Scale bar is 100 mm. doi:10.1371/journal.pone.0007626.g009

Figure 10 .
10Histological sections of ''Stygimoloch'' squamosal horn. (A-C), MOR 560.CrSp.1-L2, a squamosal horn in erosion mode in (A) longitudinal and (B,C) transverse section becomes smaller. The red arrows in (C) point to a surface that is degrading with no sign of metaplasia with a thin metaplastic veneer. Scale bar is 2 cm in (A) and 1 mm in (B) and (C). doi:10.1371/journal.pone.0007626.g010

Table 1 .
1A growth series of Pachycephalosaurus wyomingensis skulls and cranial elements from oldest to youngest.Skull
Fp dome Sq horn #1

Sq horn/node #2

Sq horn/node #3

Horn/node

Ontogenetic

Table 2 .
2A growth series of Pachycephalosaurus wyomingensis skulls and cranial elements from youngest to oldest, plus two pachycephalosaurid skulls examined in this study.Specimen
Taxon
Reference
Type Element
This study: Ontogenetic Series
Formation & State

Pachycephalosaurus wyomingensis

TCNI 2004.17.1 Dracorex hogwartsia
Bakker et al. (2006)
x
skull (cast)
juvenile
Hell Creek: SD

UCMP 119433 Stygimoloch spinifer
Galton & Sues (1983) x
squamosal
subadult
Hell Creek: MT

YPM 335
Triceratops
Marsh (1896)
squamosal
subadult
Lance: WY

Triceratops
Hatcher et al. (1907)

Pachycephalosaurus sp.
Brown & Schlaijker
(1943)

Stygimoloch spinifer
Galton & Sues (1983)

Dracorex hogwartsia
Bakker et al. (2006)

UCMP 119433 Stygimoloch spinifer
Galton & Sues (1983) x
squamosal
subadult
Hell Creek: MT

MPM 7111
Stenotholus kohleri
Giffin et al. (1987)
x
partial skull
subadult
Hell Creek: MT

Stygimoloch spinifer
Gabriel & Berghaus
(1988)

MPM 8111
Stygimoloch spinifer
Gabriel & Berghaus
(1988)

partial skull
subadult
Hell Creek: ND

UCMP 131163 Stygimoloch spinifer
Goodwin et al. (1998)
partial skull
subadult
Hell Creek: MT

UCMP 128383 Stygimoloch spinifer
Goodwin et al. (1998)
partial skull & skeleton
subadult
Hell Creek: MT

Goodwin & Horner
(2004)

AMNH 21542 Stygimoloch spinifer
Goodwin et al. (1998)
partial skull & skeleton
subadult
Hell Creek: MT

CCM 87-1
Pachycephalosaurus wyomingensis Giffin (1989)
frontoparietal dome (cast) subadult
Hell Creek: MT

MOR 560
Pachycephalosaurus wyomingensis This study
squamosal horn
subadult
Hell Creek: MT

UCMP 556078 Pachycephalosaurus wyomingensis This study
skull (cast)
subadult
Lance: SD

USNM 264304 Pachycephalosaurus wyomingensis Giffin (1989)
frontoparietal dome
adult
Hell Creek: MT

USNM 12031
Troödon wyomingensis
Gilmore (1931)
x
partial skull (lit.)
adult
Lance: WY

CM 3180
Troödon wyomingensis
Gilmore (1936)
partial skull (lit.)
adult
Lance: WY

AMNH 1696
Pachycephalosaurus grangeri
Brown & Schlaikjer
(1943)

x
skull (cast)
adult
Hell Creek: MT

Pachycephalosaurus wyomingensis Sues & Galton (1987)

DMNS 469
Pachycephalosaurus reinheimeri
Brown & Schlaijker
(1943)

x
skull
adult
Lance: SD

Pachycephalosaurus wyomingensis Sues & Galton (1987)

VRD 13
Pachycephalosaurus wyomingensis Goodwin & Horner
(2004)

skull
adult
Hell Creek: MT

UCMP 131334 Pachycephalosaurus wyomingensis This study
skull
adult
Hell Creek: MT

MOR 295
Pachycephalosauridae indet.
This study
partial skull
younger adult
Hell Creek: MT

MOR 597
Stegoceras sp.
This study
?postorbital
juvenile-subadult
Judith River: MT

Published name and citation, type status, element, ontogenetic series, formation and state from the Upper Cretaceous of the Western Interior, USA, are listed. All
formations are Maastrichtian age except for the Campanian Judith River Formation. Abbreviations: Fm, formation; lit., literature review only; MT, Montana; ND, North
Dakota; SD, South Dakota; WY, Wyoming.
doi:10.1371/journal.pone.0007626.t002

PLoS ONE | www.plosone.org
October 2009 | Volume 4 | Issue 10 | e7626
AcknowledgmentsWe thank Ellen Lamm for the preparation of histological sections. Robert
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Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis
06 Mar. 2013

Qiong Liu
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Yuheng Gong
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Yuqin Cao
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Xintian Wen
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Xiaobo Huang
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Qigui Yan
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

Yong Huang
Sanjie Cao
College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

College of Veterinary Medicine and Institute of Preventive Veterinary Medicine
Sichuan Agricultural University
China

46 Xinkang Road, Yaan cityYucheng District, Sichuan ProvinceChina

Construction and immunogenicity of a ∆apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis
06 Mar. 2013EDF2553F2540D7024EEFCFB4B523AAFB10.4102/ojvr.v80i1.519Received: 09 Sept. 2012 Accepted: 03 Dec. 2012
The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection.In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named DapxIC/ ompP2.The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis.It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice.The LD 50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD 50 of the mutant strain and parent strain in mice were determined to be 1.0 × 10 7 CFU and 3.5 × 10 5 CFU respectively).The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.

Introduction

Actinobacillus pleuropneumoniae (AP) is the causative agent of porcine pleuropneumoniae, a highly contagious disease contributing to fatalities in pig production worldwide (Sebunya & Saunders 1983).The pathogen is transmitted by air or direct contact with infected pigs (Chiers et al. 2002).Haemophilus parasuis is a commensal bacterium in the upper respiratory tract of conventional pigs, but under appropriate conditions, can invade and cause severe systemic disease (Glasser`s disease) (Hoefling 1991).Both bacteria are members of the family of Pasteurellaceae; their growth requires a V factor (NAD), but no X factor (hemin).In recent years, the porcine industry has suffered a significant increase in the incidence, morbidity and mortality associated with Glasser`s disease (Oliveira & Pijoan 2004) an infection often combined with AP, which has caused serious economic loss world-wide.

The Apx is characterised as one of the major virulence determinants of A. pleuropneumoniae (Frey & Nicolet 1990).Up till now, four different Apx toxins (ApxI to ApxIV) have been described and varying combinations of Apx toxins are produced in different A. pleuropneumoniae serovars.For instance, serovars 1, 5, 9 and 11 generate ApxI and ApxII (Tascon et al. 1993).Amongst the Apx toxins, ApxI, which is secreted by the most virulent serovars such as serovars 1, 5, 9, 10 and 11, has shown strong haemolytic activity and cytotoxic activity (Kamp et al. 1991).Meanwhile, ApxI, ApxII and ApxIII are strongly immunogenic and involved in the induction of protective immunity.The toxin ApxI is encoded on polycistronic operons apxICABD (Frey et al. 1994).In these operons, the A gene encodes the protoxin, whilst the C gene encodes a cyltransferase, which is involved in the post-translational activation of the protoxin in the cytoplasm (Issartel, Koronakis & Hughes 1991).The B and D genes respectively encode an ATPase and a protein adaptator to facilitate Apx toxin secretion (Thanabalu et al. 1998).

The inactivated whole-bacteria vaccine has been demonstrated to elicit protection against infection from the same serovar of A. pleuropneumoniae (Hensel et al. 2000) and subunit vaccines have been tested to provide some immunity with reduction in clinical symptoms and pulmonary lesions (Goethe et al. 2000).However, in terms of inactivated whole-bacteria vaccine, it was not able to generate cross protection to infection from heterologous serovars or to provide long-lasting immunity.Secondly, in terms of subunit vaccines, although the subunit vaccines contained the mixed outer membrane proteins and although proteins secreted from several serovars or recombinant toxins have been shown to protect animals from clinical infection (Maas et al. 2006), the relatively higher production cost of mixing different antigens would undoubtedly limit their application.In contrast, pigs surviving infection with one serovar are at least partially protected against infection by other serovars.Consequently, developing a live vaccine by attenuation of A. pleuropneumoniae has become a dominant research field.

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The outer membrane protein P2 (ompP2) of H. parasuis is encoded by the ompP2 gene and is an immunodominant porin with considerable antigenic heterogeneity amongst different serovars of H. parasuis (Mullins et al. 2009).The ompP2 has been targeted as a potential vaccine candidate.As a porin, ompP2 plays a role in colonisation and has been shown binding to specific components of human mucin (Reddy et al. 1996).It was demonstrated that ompP2 could be expressed in H. parasuis and that it shows good antigenicity.

Inactivated bacterial vaccines and subunit vaccines are currently the main ways of preventing porcine pleuropneumoniae.However, the multitude of serovars (15 serovars) and the limited cross-protection conferred by bacterins or Apx toxin-based subunit vaccines (Jolie, Mulks & Thacker 1995) makes treatment of A. pleuropneumoniae infection difficult.A live vaccine is considered to offer the best prospects of obtaining cross-protection against this bacterium.In the current study, an attenuated mutant A. pleuropneumoniae strain with the ompP2 gene of H. parasuis serovar 5 (Nagasaki) (Takahashi et al. 2001) was developed as a potential vaccine candidate without introducing an antibiotic resistance marker by using a sucrose counterselection strategy (Oswald et al. 1999).Actinobacillus pleuropneumoniae serovar 5 (SC-1), is one of the predominant serovars in China, and produces both apxI and apxII toxins.The apxIC region was targeted for disruption with a gene of H. parasuis to produce a mutant strain that secretes an inactive toxin with full antigenic properties.Here, we describe the construction of the homologous recombination vector, the characterisation of the resultant mutant strain and the testing of its virulence and protective efficacy against A. pleuropneumoniae and H. parasuis infection in mice.

Materials and methods

Bacteria isolates and growth conditions

Haemophilus parasuis serovar 5 (Nagasaki) and A. pleuropneumoniae serovar 5 (SC-1) were selected for this study.The bacterial strains were both grown on tryptic soy agar (TSA, DifcoTM, BD) and in tryptic soy broth (TSB, DifcoTM, BD) supplemented with 1% (wt/vol) NAD.All bacterial strains were grown at 37 °C.

Escherichia coli DH5α was routinely maintained in Luria-Bertani medium (LB) or maintained in LB supplemented with 100 mg/mL of ampicillin or 50 mg/mL of kanamycin when containing the relevant vector.

The vector pBOSKDIC, constructed by Dr Guobin Wang, was used for constructing the vector of the homologous recombination.

Construction of vector for homologous recombination

The first step was to transform the restriction site on the right homologous arm of homologous recombination vector pBOSKDIC with SpeI and MluI.The primers used in this study are listed in Table 1.A PCR of the right homologous arm was conducted by initial denaturing at 94 °C for 5 min, followed by 30 cycles of denaturing at 94 °C for 45 sec, annealing at 55 °C for 30 sec and extension at 72 °C for 1.5 min, with a final extension at 72 °C for 10 min.The reconstructive vector was called pBOSKDIC-1.The ompP2 gene of H. parasuis was then amplified by PCR with the same PCR conditions as described above, The amplicons of ompP2 were checked in agarose gel electrophoresis, purified and cloned in pMDsimple-19T vector (TaKaRa Biotechnology [Dalian] Co., Ltd), followed by subcloning between SpeI and MluI sites of the reconstructive vector pBOSKDIC-1.The vector was use for homologous recombination and called pBOSKDapxIC/ ompP2 (Figure 1).

Electroporation of Actinobacillus pleuropneumoniae and selection of recombinants

The transfer vector pBOSKDapxIC/ompP2 was extracted by TM Vector Mini Kit II (OMEGA).The competent cells of A. pleuropneumoniae serovar 5 (SC-1) were prepared according to the general optimisation protocol for the transformation of bacteria provided by the Electroporation System (Gene Pulser Xcell TM , BIO-RAD).Pulse conditions were as follows: cuvette 0.2 cm-diameter; voltage 2.5 kV; capacitance 25 uF; resistance 800 Ω; time constant 5 ms.After electroporation, the cells were transferred into 1 mL TSB supplemented with NAD and incubated immediately at 37 °C, at 180 r/min for 3 h.The recovered cells were plated onto the TSA plates, supplemented with 1% (wt/vol) NAD and 50 ug/mL of kanamycin, incubated overnight at 37 °C.Kanamycin resistant colonies were selected and inoculated onto TSA-sucrose plates, which were incubated at 37 °C to confirm that the sucrose sensitivity was conferred by transfer of the vector (Figure 1).

Identification

The genomic DNA of the parent and mutant strains was extracted individually.Primer pairs IC-F and IC-R were used to amplify the relevant fragments from the genomic DNA of the parent and mutant strains.The DNA fragments of these strains were separated on a 1% agarose gel.

Growth and genetic stability of the mutant strain

The mutant strain was inoculated into TSB supplemented with 1% (wt/vol) NAD, The bacterial concentration was determined at OD600 every hour, and the value recorded.The growth curves were drawn to compare features of growth cycles.

The genetic stability of the inserted ompP2 gene in the genome of the mutant strain was tested.This was done by propagating the mutant strain for 10 passages serially in TSB supplemented with 1% (wt/vol) NAD, and then amplifying the relevant fragments by PCR from the colony of each passage.

Haemolytic activity and NAD dependency test

The haemolytic activity and NAD dependency of the parent and mutant strains were examined.Briefly, they were inoculated onto sheep blood agar plates overnight at 37 °C.Escherichia coli DH5α was used as negative control.The haemolytic activity of the strains was recorded.At the same time, the growth was observed of the mutant strain inoculated in the TSB, supplemented with 1% (wt/vol) NAD and incubated overnight at 37 °C.

Safety and vaccination efficacy of the mutant strain in mice

Cultures of A. pleuropneumoniae serovar 5 were grown overnight at 37 °C in TSB supplemented with NAD, followed by dilution to 1:1000.The diluted culture was incubated again until the OD600 reached 0.8.At this point, the viable count of A. pleuropneumoniae DapxIC/ompP2 was found to three concentrations (Table 2).For the virulence test, three groups of eight 6-week-old female Balb/C mice (Sichuan Province Huaxi Experimental Animal Centre, Chengdu, China) were injected intraperitoneally (i.p.) with 200 µL TSB medium containing various bacterial counts of A. pleuropneumoniae or H. parasuis.Survival of the mice was recorded after 72 h (Table 2).The lethal dose of 50% (LD 50 ) value of the mutant strain DapxIC/ompP2 was calculated by Karber's method.

Serological measurement

Serum was isolated from whole blood collected from mice at four points in time: before vaccination (day 0), before the secondary vaccination (day 14), before the third vaccination (day 28) and before challenge (day 42).Inactivated A. pleuropneumoniae and H. parasuis were used as a somatic antigen by ELISA.Briefly, the 96-well Enzymelinked immunosorbent assay (ELISA) microplates were coated overnight at 4 °C with 0.08 mg/mL of inactivated A. pleuropneumoniae and H. parasuis diluted in 100 mL of 50 mM sodium carbonate buffer, pH 9.6.Plates were washed three times with washing buffer (0.05% Tween 20 in PBS, PBST) and blocked for 1 h at 37 °C with blocking buffer (1% BSA in PBST).After three washes, serum samples were diluted to 1:100 in blocking buffer, added to each well (100 µL per well) in duplicate and incubated for 1 h at 37 °C.After three washes, 100 µL of HRP-conjugated goat antis mice IgG (SBA, America), diluted to 1:5000 in blocking buffer, was added to each well and incubated at 37 °C for 1 h.After three washes, 50 µL TMB was added to each well at room temperature in dark reaction for 15 min The reaction was then stopped by adding 100 µL of 2 M H 2 SO 4 per well.The absorbance was read at 450 AA with an ELISA micoplate reader (MODEL 680, BIO-RAD).

Results

Identification of Actinobacillus pleuropneumoniae apxIC mutant lacking antibiotic resistance markers

After electroporation of A. pleuropneumoniae competent cells with the vector pBOSKDapxIC/ompP2, some transformants   were screened from growth on TSA plates containing kanamycin and sucrose, respectively.Three transformants with the phenotype of Kan r and Suc s were chosen for propagation in TSB medium without kanamycin or sucrose to promote the second order recombination event and excised the kanamycin resistance and sacB genes.Finally, the cells were further grown overnight in TSB-NAD in the absence of kanamycin, then grown onto TSA-Sucrose plates.The Suc r colonies were replica plated onto TSA-Sucrose and TSA-Sucrose-Kan r plates to choose the colonies with the Kan s and Suc r phenotype.

Strains

The ApxIC gene of A. pleuropneumoniae was identified by PCR (Figure 2), the genomic DNA of the parental strain had an amplicon of about 800 bp and the mutant stain an amplicon about 1400 bp.The amplification of the ompP2 gene of H. parasuis yielded a band of 1100 bp.This suggests that the ompP2 gene of H. parasuis was inserted into the ApxIC gene of A. pleuropneumoniae at the deletion site.

Growth and genetic stability of the mutant strain

No obvious difference was observed in vitro growth curves of the parental strain and mutant strains, indicating that the ompP2 gene of H. parasuis that took the place of the apxIC gene had no significant influence on the growth of A. pleuropneumoniae (Figure 3).

A PCR fragment with a size of 1400 bp was amplified from all ten serial passages in TSB supplemented with 1% (wt/vol) NAD, indicating that the ompP2 gene of H. parasuis was stably inserted into the genome of the mutant strain, and the apxIC gene of A. pleuropneumoniae had been deleted (Figure 4).

Haemolytic activity test

The functional activity of the ApxI toxin produced by DapxIC/ompP2 was examined in the haemolytic assay.Fully active ApxI toxin had haemolytic activity, as demonstrated by clear zones surrounding the colonies of A. pleuropneumoniae on sheep blood agar (Figure 5, Section P).The apxIC gene of A. pleuropneumoniae was deleted, and the ompP2 gene of H. parasuis showed no haemolytic activity, thus the mutant strain was lacking haemolytic activity.This was evident from the absence of a clear zone surrounding the colonies of the A. pleuropneumoniae mutant on sheep-blood agar (Figure 5, Section M).

Safety of the mutant strain in mice

To test the virulence of the mutant strain, various concentrations of A. pleuropneumoniae DapxIC/ompP2 and serovar 5 of H. parasuis and A. pleuropneumoniae parent strain were prepared and administrated to mice via the intraperitoneal route.No deaths were observed in mice that received 2.0 × 10 6 CFU of H. parasuis and 8.0 × 10 6 CFU of DapxIC/ompP2, whilst 100% mortality was recorded in mice that received the parent strain at doses of 2.0 × 10 6 CFU.Therefore, compared to the parent strain  The mutant strain

The parental strain

The mutant strain and serovar 5 of H. parasuis, virulence of the mutant was attenuated at least 30-fold (Table 2).

Serological measurement

Serum was isolated from whole blood collected from mice at four points in time: before vaccination (day 0), before the  secondary vaccination (day 14), before the third vaccination (day 28) and before challenge (day 42).Antibodies against the somatic antigen of inactivated A. pleuropneumoniae and H. parasuis were examined using ELISA (see Figure 6).The ELISA titres were defined as the highest serum dilution resulting in OD450 twice and the negative control serum at a dilution of 1:100.

Discussion

It is known that ApxI toxin is a very important member of the RTX family and common structural features are present in the RTX toxins.When used as a subunit vaccine, it could offer a good level of protection in mice against A. pleuropneumoniae infection (Seah, Frey & Kwang 2002).

The ApxIC contains 13 repeated nonapeptides and plays an important role during the interaction between ApxI toxins (Issartel et al. 1991).In earlier studies, expression of the apxC gene was generally inactivated by insertion of foreign marker genes or by deletion of the gene (Liu et al. 2007).

These mutant strains have the potential to reverse virulence by releasing the inserted genes and transmitting the marked antibiotic resistance gene to the animal's normal bacterial flora.Live vaccines are considered to offer the best prospect for obtaining cross-protection against A. pleuropneumoniae serovar 5 and H. parasuis serovar 5.In 1999, Oswald constructed an A. pleuropneumoniae UreaC mutant strain using a new method, the sucrose counter-selectable system, but its application in A. pleuropneumoniae has been limited so far (Oswald et al. 1999).The method demonstrated that it is possible to modify the A. pleuropneumoniae genome without inserting an antibiotic resistance marker.

Based on these previous studies, we constructed a live attenuated ApxI inactivation mutant strain by deleting the ApxIC toxin, and inserting the ompP2 gene of H. parasuis.

The A. pleuropneumoniae serovar 5 (SC-1) was selected as the parent strain for the mutant construction, because it is one of the most prevalent serovars in China, and is capable of secreting ApxI and ApxII.

The results indicate that the mutant strain DapxIC/ ompP2 is capable of secreting ApxI, ApxII and ompP2 of H. parasuis after deletion of apxIC as the DapxIC/ompP2 still contains apxIB and apxID genes, which encode proteins that are essential for the secretion of the toxin (Welch 1991).

Compared with the parent strain, virulence of the mutant strain (DapxIC/ompP2) was attenuated 30-fold, providing more evidence that the ApxI of A. pleuropneumoniae serovar 5 is necessary for full virulence (Goethe et al. 2000).

In terms of haemolytic activity, the mutant strain had lower visible haemolytic activity on agar plates.It was proved that apxIC gene was absence from the genome of A. pleuropneumoniae (SC-1).The serological measurement of mice serum show that the mutant strain provided lower protection against H. parasuis than A. pleuropneumoniae.This is possibly because A. pleuropneumoniae is the parent strain, whilst the expression of ompP2 gene in the mutant strain is not enough to protect against invasion of H. parasuis.Another aspect is that the ompP2 gene is not the most important antigen of H. parasuis, but the mutant strain could still protect mice against H. parasuis and A. pleuropneumoniae.

In summary, the mutant strain DapxIC/ompP2 had greatly reduced virulence.It needs to be pointed out that the mouse model is only a preliminary assay and full studies in pigs are needed to test this mutant in future.

Conclusion

In this study, we successfully constructed a mutant strain of A. pleuropneumoniae (SC-1) named DapxIC/ompP2, whilch can provide the protection against H. parasuis and A. pleuropneumoniae.

homologous arm; R, right homologous arm.

FIGURE 1 :ABLE 2 :
12
FIGURE 1: Schematic representation of the construction of the Actinobacillus pleuropneumoniae mutant strain.

Marker III; (a) lane 1, the parent strains; lane 2, the first single cross-over mutants; (b) lane 1, the parent strains; lane 2, the mutant strain; lane 3, polymerase chain reaction amplification using primer pair ompP2-F and ompP2-F.

FIGURE 2 :FIGURE 3 :
23
FIGURE 2: Confirmation of apxIC gene disruption in the genome of the mutant strain by polymerase chain reaction amplification using primer pair IC-F and IC-R.

DNA marker III; lanes 1-10(a), apxIC gene fragment amplified from the mutant strain of 1-10 continuous passages; lane 1(b), apxIC gene fragment amplified from the parent strain.

FIGURE 4 :
4
FIGURE 4: Stability of the inserted ompP2 gene in the genome of the mutant strain over 10 passages by polymerase chain reaction.

P

FIGURE 5 :
5
FIGURE 5: Identification of haemolysis of mutant strain.

FIGURE 6 :
6
FIGURE 6: Indirect ELISA analysis of the IgG antibody in murine serum after immunisation.Six-week-old BALB/c mice were subcutaneous injected three times every two weeks with the mutant strain at a dosage of 3.0 × 10 6 CFU (group A and B) 200 μL per mice.The mice of group B and D were vaccinated with 7.0 × 10 5 CFU of the parent strain (serovar 5 of Actinobacillus pleuropneumoniae), using inactivated bacteria as a coating antigen.Absorbance was measured at 450 nm in an automated plate reader.Results are expressed as means of the OD450 ± s.d.(n = 3).Numbers on the x-axis indicate week post-primary immunisation.

http://www.ojvr.org
AcknowledgementsThis work was supported by the Science Expertise of Public Interest Career -Agriculture (No.201303034).Competing interestsThe authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article.Authors' contributions
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Identification of haemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies. E M Kamp, J K Popma, J Anakotta, M A Smits, Infection and Immunity. 591991

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Development of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae infection. A Maas, J Meens, N Baltes, I Hennig-Pauka, G F Gerlach, 10.1016/j.vaccine.2006.06.047Vaccine. 242006

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S Oliveira, C Pijoan, 10.1016/j.vetmic.2003.12.001Haemophilus parasuis: New trends on diagnosis, epidemiology and control. 200499

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker. W Oswald, W Tonpitak, G Ohrt, G Gerlach, 10.1111/j.1574-6968.1999.tb08721.xFEMS Microbiology Letters. 1791999

Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyngeal mucin. M S Reddy, J M Bernstein, T F Murphy, H S Faden, Infection and Immunity. 641996

The N-terminal domain of RTX toxin ApxI of Actinobacillus pleuropneumoniae elicits protective immunity in mice. J N Seah, J Frey, J Kwang, 10.1128/IAI.70.11.6464-6467Infection and Immunity. 702002. 2002

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Measurement of water-use by Jatropha curcas L. using the heat-pulse velocity technique
Aug Sep Oct Nov Dec Jan Feb Total

M B Gush firstname.lastname@example.com
CSIR, ℅ Agrometeorology
School of Environmental Sciences
University of KwaZulu-Natal
Private Bag X013209ScottsvilleSouth Africa

Measurement of water-use by Jatropha curcas L. using the heat-pulse velocity technique
1816-7950 = Water SA (on-line)Aug Sep Oct Nov Dec Jan Feb TotalD66C0AC88AC94D8EB3B34324F4F821AFheat ratio methodsap flowtranspirationwater resource impacts
In response to the proposed introduction of the potential bio-diesel species Jatropha curcas (Linnaeus) to South Africa, field experiments were conducted to investigate its likely water-use impacts relative to other forms of vegetative land use.As no existing water-use data could be found for this species worldwide, sap flow in Jatropha curcas trees was measured continuously for a 17-month period at two sites in eastern South Africa.These consisted of young (4-year-old) trees at a relatively wet site and mature (12-year-old) trees at a dry site.The heat-ratio method of the heat-pulse technique was utilised, together with measurements of meteorological variables and soil water.Sap-flow rates varied according to tree age, season, prevailing meteorological conditions, and soil moisture levels.Peak sapflow rates occurred during the warm wet summer months, but due to the deciduous nature of the species, water use was negligible during winter.Scaled-up sap-flow measurements resulted in estimates of total annual transpiration of 1 983 ℓ (147 mm) for a 4-year-old J. curcas tree, and 4 884 ℓ (362 mm) for a 12-year-old J. curcas tree.The study concluded that the J. curcas trees studied were conservative in their water use, and were unlikely to transpire more water than indigenous vegetation types of the area.

Introduction

In response to rising oil prices and the quest for alternative, economically viable and environmentally sustainable forms of energy, certain plant species with bio-energy potential have been proposed for large-scale planting and bio-fuel production.International interest in Jatropha curcas as a drought-tolerant, fastgrowing, renewable bio-energy crop has grown significantly in recent years.Countries such as India have initiated large-scale plantings of J. curcas in efforts towards the increased use of bio-diesel as an alternative to fossil fuel imports (Francis et al., 2005).In South Africa, the government has received numerous requests for permission to plant this species, but, apart from certain trial plantings, has imposed a moratorium on commercial plantings.This has been due to, amongst other considerations, the extremely limited data available on its potential environmental impacts (specifically water-use).This knowledge gap is of particular concern in a dry country such as South Africa, where evapotranspiration from vegetation is the component of the water balance that accounts for the greatest loss of water from catchments.Accurate estimates of water-use are therefore fundamental to gaining a good understanding of the hydrological impacts of a specific plant species or vegetation type.Where large-scale changes in vegetation cover are being proposed, this aspect becomes particularly important because the difference in evapotranspiration between the current and the proposed vegetation will be directly related to changes in available water in that catchment (ultimately streamflow).Consequently, large-scale changes in land use could have significant hydrological implications if the water use of the introduced species were significantly different to that of the vegetation it would replace.

In this study, sap-flow measurements of young (4-year-old) and mature (12-year-old) Jatropha curcas trees were conducted to assess the water-use trends of this species relative to other forms of vegetative land use.Plant sap flow (transpiration) was measured using the heat-pulse technique, together with measurements of meteorological variables and soil moisture at two sites in KwaZulu-Natal Province, South Africa (Gush and Moodley, 2007).

Materials and methods

Site selection

Two monitoring sites were selected along the subtropical eastern seaboard of South Africa within the KwaZulu-Natal (KZN) Province (Fig. 1).These sites consisted of:

Sap-flow measurements

The heat-pulse velocity (HPV) technique is recognised internationally as an accepted method for the measurement of sap flow in woody plants and has been extensively applied in South Africa (Dye and Olbrich, 1993;Dye, 1996;Dye et al., 1996).The heat ratio method (HRM) of the HPV technique (Burgess et al., 2001) was selected because of its ability to measure low rates of sap flow, often characteristic of plants growing under dry conditions typically experienced in South Africa.Sap-flow monitoring systems were installed in two trees at each site, using thermocouple pairs and heater probes inserted to different depths within the sapwood to determine radial variations in sap flow.Due to the fast-growing nature of Jatropha curcas the probes were completely removed and repositioned to their correct depths at the start of the growing season (October), and then rechecked periodically during the course of the monitoring period.A CR10X data-logger connected to two AM16/32 multiplexers (Campbell Scientific, Logan, UT) was programmed to initiate the heat pulses and record hourly data from the respective thermocouple pairs.Cellular phone modems connected to the loggers allowed remote downloading of data as well as uploading of programmes to the logger.

Sap-flow data analysis

Once sap-flow monitoring had been completed, additional supplementary information required to complete the HPV data analysis was collected.All meteorological data were aggregated into daily, monthly and annual totals.Measurements of sapwood area, sapwood moisture content, wood density and the width of wounded (non-functional) xylem around the thermocouples ('wound widths') were conducted on the trees.The trees could not be destructively sampled as they constituted an agricultural research trial, so wood samples and tree cores were taken to determine these variables.

Measured heat-pulse velocities were corrected for sapwood wounding caused during the drilling procedure, using woundcorrection coefficients described by Swanson and Whitfield (1981).The corrected heat-pulse velocities were then converted to sap-flux densities according to the method described by Marshall (1958).Finally, the sap-flux densities were converted to whole-tree total sap flow by calculating the sum of the products of sap-flux density and cross-sectional area for individual tree stem annuli (determined by below-bark individual probe insertion depths and sap-wood depth).Hourly sap-flow values were aggregated into daily, monthly and annual totals for both trees at each site.

Results

Meteorological variables

Monthly variations in mean maximum and minimum temperature, total rainfall and total solar radiation measured at OSCA and Makhathini are illustrated in Figs. 2 and 3 respectively.

Although average maximum and minimum air temperatures at both sites declined from April onwards, they never dropped below 24.4°C and 9°C respectively.The lowest recorded temperature for the entire monitoring period was 2.3°C on 17 June  (Lynch and Schulze, 2006) and these dry conditions were a consideration in the interpretation of the results of this study.Reference evapotranspiration (ET o ), as calculated using the FAO56 method (Allen et al., 2004), was 1 251.7 mm for the OSCA site and 1 310.7 mm for the Makhathini site (Table 1).

Daily values ranged from a maximum of approximately 8 mm in summer to 1 mm in winter at both sites.

Sap flow

Over a calendar year (March 2005 to February 2006) the sapflow totals for the sample trees at OSCA were 1 899 kg (ℓ) and 1 983 kg for Trees 1 and 2. Individual tree sap-flow values were scaled up to represent a larger area (i.e. a J. curcas plantation).

Tree spacing at the OSCA study site (3 m by 4.5 m) translated into a planting density of 740 stems per hectare (spha).By scaling the individual tree sap-flow measurements up to a hectare, and converting to mm-equivalent, sap-flow (transpiration) totals were calculated to be 140.6 mm and 146.9 mm for Trees 1 and 2. A similar exercise was conducted for the Makhathini site.Some assumptions were required because the sample trees consisted of just a single row of 10 trees along the homestead fence.These trees were spaced approximately 3 m from each other (as at OSCA), and so a similar planting density to the OSCA site was assumed (740 spha) despite the absence of additional rows of trees.Consequently it is possible that the scaled-up sap-flow rates calculated here are slightly higher than would be experienced in a plantation environment, as a result of reduced competition for light, water and nutrients.The annual sap-flow totals for the Makhathini trees were 4 884 kg and 4 036 kg for Trees 1 and 2, which translated into totals of 361.8 mm and 298.9 mm for Trees 1 and 2, given the planting density.Daily fluctuations in sap-flow (transpiration) (kg•d -1 ) for the trees at the OSCA and Makhathini sites are shown in Figs. 4 and 5.

Discussion and conclusions

Sap-flow rates in the J. curcas trees were season-dependent due largely to the deciduous nature of the species, although day length and rainfall had an effect on the seasonal pattern (Figs. 4 and 5).Daily sap-flow totals fluctuated widely due to changes in energy availability (solar radiation) and VPD, but also    potentially due to the timing and amount of individual rainfall events, and their replenishing effect on soil moisture availability.The highest daily sap-flow totals recorded at the OSCA site were 30.3 kg•d -1 (Tree 1) and 32.9 kg•d -1 (Tree 2), while daily averages over a full year (March 2005 -February 2006) were 5.2 kg•d -1 and 5.4 kg•d -1 respectively.Maximum sap-flow rates in summer contrasted with a cessation of sap flow in the cooler and drier winter months.The declining sap flows observed closer to the winter months, resulted from a combination of meteorological conditions (lower temperatures and reduced solar radiation), changing tree physiological conditions (leaf drop) and declining soil-water content.An increase in sap flow after winter dormancy coincided closely with the first good rains, the development of new leaves and the onset of warmer/wetter conditions in mid-to late October.Figure 6 illustrates seasonal changes in heat-pulse velocities recorded in the J. curcas trees at the OSCA site in relation to changing meteorological conditions (solar radiation and rainfall) and soil water contents (average of 3 sample depths).Three periods are illustrated during which significant changes in sap flow were observed, namely transition periods at the onset of winter dormancy (June 2005), at the start of the summer growing season (Oct/Nov 2005), and at a corresponding period during the following winter (June 2006).After winter dormancy the first flush of new leaves occurred soon after the onset of warmer, wetter conditions early in October 2005.The resumption of sap flow was detected from 16 October 2005 onwards.Higher (although more variable) solar radiation and soil-water contents characterised this summer period.Sap flow on days of significant rainfall (e.g.5/6 November 2005 -37.4 mm) was low due to energy constraints resulting from reduced solar radiation because of clouds, low atmospheric VPD and wet leaves.However, within a day or two thereafter responses to the additional soil water provided by these events were manifested through increased sap-flow rates.Sap flow towards the end of the second growing season (June 2006) was sustained for longer than during the preceding year and appears to be the result of greater soil-water availability.

J. curcas sap-flow rates at the Makhathini site appeared to be highly rainfall-dependent (Fig. 5).This is possibly as a result of the sandy soils found in this area, which would have rapid drainage and a low capacity to retain water near the surface where the bulk of the tree roots are.Consequently, individual rainfall events initiated rapid responses in sap flow as the trees made use of the readily available soil moisture.However, as the water drained or evaporated from the sandy soils, so the sapflow rates declined markedly, until the next rainfall event.This trend was most dramatic at the start of the hydrological year (October -January), but became less so towards the end (February -July).The highest daily sap-flow totals recorded at the Makhathini site were 79.6 kg•d -1 (Tree 1) and 55.5 kg•d -1 (Tree 2), while daily averages over a calendar year (March 2005 to February 2006) were 13.4 kg•d -1 (Tree 1) and 11.1 kg•d -1 (Tree 2).Tree 1 at Makhathini was observed to transpire more water than Tree 2. When the probes were removed from the trees at the end of the monitoring period, it was noticed that Tree 2 had an area of dead wood in one part of the stem, and it is possible that this had an impact on the overall water-use of that tree and the estimation of whole-tree sap flow.

Although the HPV data for the Makhathini site showed similar seasonal patterns to that of the OSCA site, there were some noticeable differences between the two sites.Firstly, the Makhathini HPV data declined far more dramatically and significantly earlier in the winter season.While the 4-year-old trees at OSCA continued transpiring until mid-June 2005, the older Makhathini trees showed no signs of sap flow after the 15 th of May 2005.Periods of water stress as early as mid-March and during the second half of April (characterised by decreasing sap flow) were alleviated by individual rainfall events.The differences in total water use between the J. curcas trees at OSCA and those at Makhathini could not be attributed to prevailing climatic conditions alone, as monthly values of reference evapotranspiration (ET o ) were similar for the two sites.Although the OSCA trees had noticeably higher sap-flow rates, presumably due to their young age and increased vigour; in terms of total volumetric water-use they were surpassed by the older and larger Makhathini trees with a greater leaf area.

It should be borne in mind that these results were obtained under conditions of below-average rainfall, especially at the OSCA site, where rainfall for the year was just 61% of the annual mean.Under these conditions and at dry sites such as Makhathini, annual sap flow will be low and constrained by low available water and the deciduous nature of the species.Nevertheless, at a higher rainfall site, the deciduous habit is still going to constrain annual evapotranspiration.From the observed results it was concluded that J. curcas trees are conservative in their water use and comparable to that of indigenous vegetation types which exhibit seasonal senescence (e.g.grassland), and significantly less than evergreen vegetation that is capable of transpiring year-round (e.g.exotic plantation forestry species).

February 2005 until 27 th June 2006.The weather station was located approximately 2 m from the trees.Sensors were mounted 2 m above the ground, and variables were continuously measured at 10 s intervals, and averaged or totalled at hourly intervals.Continuous measurements of soil-water content were carried out at the OSCA site using CS616 probes (Campbell Scientific, Logan, UT).No soil-moisture measurements were taken at the Makhathini site.The Makhathini site consisted of mature J. curcas trees situated along the fence-line of a rural homestead in the Makhathini flats region of Northern KZN.The site (grid reference S 27º 24' 06.9'; E 32º 11' 48.6' and altitude 75.2 m a.m.s.l.) was nonriparian and was situated approximately 20 km east of Jozini, KZN Province, close to the Makhathini Agricultural Research Station.Based on anecdotal evidence from local inhabitants the trees were estimated to be approximately 12 years old (planted January 1994).The trees were approximately 6 m tall with an LAI of 2.26 measured in mid-December 2005.Sap flow and selected meteorological variables (temperature, relative humidity and solar radiation) were measured hourly at the Makhathini site for a 16-month period from 5 th March 2005 until 2 nd July 2006.

Figure 2 Figure 3
23
Figure 2 Monthly values of meteorological variables recorded between February 2005 and June 2006 at the OSCA site

Figure 4
4
Figure 4 Daily sap-flow totals (kg•d -1 ) for two 4-year-old Jatropha curcas trees, and rainfall at the OSCA site, between February 2005 and June 2006

Figure 5
5

• 4-year-old Jatropha curcas trees at the Owen Sithole College of Agriculture near Empangeni • 12-year-old Jatropha curcas trees at a homestead in the Makhathini flats, northern KZN.
toring commenced on 27 January 2005. Leaf area index (LAI)measurements taken with a Li-COR LI-2000 Plant Canopy Ana-lyser in mid-December 2005 (when leaf area was at a maximum)gave an LAI of 1.28 for the trees. Continuous measurements ofsap flow (transpiration) and meteorological variables (rainfall,solar radiation, air temperature, relative humidity, wind speedand wind direction) were taken hourly for a 17-month periodfrom 2 ndThe Owen Sithole College of Agriculture (OSCA) is locatedapproximately 20 km north-west of Empangeni, in the KZNprovince of South Africa. The identified monitoring site (gridreference S 28º 38' 36.7'; E 31º 55' 36' and altitude 44.2 ma.m.s.l.) consisted of a J. curcas trial planted in January 2002.It was a valley-bottom site, close to a stream but non-riparian,being approximately 30 m from the stream channel. The sitewas maintained under controlled conditions (weed control, butno irrigation), and consisted of 24 J. curcas trees planted in two
blocks of 6X2 trees.Spacing was 4.5 m (between rows) by 3.0 m (within rows).The trees were 2.5 m tall, had stem diameters of approximately 0.1 m at a stem height of 0.15 m when moni-Available on website http://www.wrc.org.zaISSN 0378-4738 = Water SA Vol.34 No. 5 October 2008 ISSN 1816-7950 = Water SA (on-line) 2

Wind speeds were generally low, averaging 1.3 m•s -1 at the OSCA site and 1.2 m•s -1 at the Makhathini site.Rainfall was significantly more variable.For the same calendar year used to calculate transpiration totals (March 2005 to February 2006) rainfall amounted to just 618 mm at OSCA and 440 mm at Makhathini.These totals were significantly below the long-term means of 1 016 mm for OSCA and 582 mm for Makhathini
Figure 1Location of the Jatropha curcasstudy sites
Available on website http://www.wrc.org.zaISSN 0378-4738 = Water SA Vol.34 No. 5 October 2008 ISSN 1816-7950 = Water SA (on-line) 3 2005 at OSCA (5.3°C on 17 June 2005 at Makhathini), while the highest recorded temperature was 41.3°C on 2 November 2005 at OSCA (41.0°C on 2 October 2005 at Makhathini).Mean annual temperature was 22.5°C for OSCA and 23.4°C for Makhathini, and average daily solar radiation over the entire period was 15.2 MJ•m -2 •d -1 at OSCA and 16.1 MJ•m -2 •d -1 at Makhathini.These warm and sunny conditions reflected the hot and dry sub-tropical climate of the study sites, providing suitable growing conditions for J. curcas.

TABLE 1 Monthly reference evapotranspiration totals (ET o ) at the OSCA and Makhathini sites
1OSCA118.4123.6112.884.972.860.365.099.8117.9124.7133.3137.8 1251.2Makh.134.9130.4104.191.082.368.071.592.3124.8132.1134.9144.4 1310.7
AcknowledgementsProject funding from the Water Research Commission of South Africa is gratefully acknowledged.Staff from the KwaZulu-Natal Dept. of Agriculture and Environmental Affairs (DAEA), specifically Mr. Francois du Toit, Mr. Jabulani Mdluli, Mr. Ben Koranteng and Mr. Diliza Masinga are thanked for assistance with site selection and maintenance.Appreciation is also extended to the Sithole family, for allowing the monitoring of Jatropha curcas trees at their homestead in the Makhathini area; and to colleagues Prof. Colin Everson, Dr. Mags Moodley, Mr. Alistair Clulow and Mr. Joshua Xaba for technical advice and assistance in the field.
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