First evidence of Kunjin strain of West Nile virus associated with saltwater crocodile (Crocodylus porosus) skin lesions.

Recently, the Kunjin strain of West Nile virus (WNVKUN ) has been detected using qRT-PCR in belly skin lesions of farmed juvenile saltwater crocodiles. This follows an established association between similar lesions and West Nile virus in American alligators. The lesions present as cutaneous lymphohistiocytic aggregates in the dermal layers of both species. While these lesion do not create an obvious defect on the live crocodile, upon tanning the lesion area collapses and does not uptake the dye evenly, thus reducing its aesthetic appeal. As a result, skins are being rejected jeopardising the economic viability of the Australian crocodile industry. Over 50 skin lesions have since been confirmed as WNVKUN -positive and preliminary evidence of lesion restructuring is presented. Horizontal transmission of WNVKUN by mosquitoes is well-established but other transmission routes, such as ingestion and cloacal shedding, need further evaluation. An infection trial is currently underway to ensure WNVKUN is the causative agent of these skin lesions.

Genetics and infection dynamics of Paratrichosoma sp in farmed saltwater crocodiles (Crocodylus porosus).

Paratrichosoma-associated helminthiasis has been identified in saltwater crocodiles under intensive farming conditions. The development of sustainable integrated management practices is dependent on a detailed understanding of Paratrichosoma population genetics and infection dynamics. This study investigated the genetic relationships of Paratrichosoma sp in a population of commercially farmed saltwater crocodiles, Crocodylus porosus, in northern Australia. 18S ribosomal RNA gene sequence data were obtained from Paratrichosoma sp eggs present in the epidermis of infected animals. A high level of genetic diversity was distributed within the Paratrichosoma sp population (241 variable positions in the 1094 bp alignment), indicating an accelerated rate of nucleotide base-pair substitutions in this genus of nematodes. Several possible environmental correlates of the incidence and intensity of helminthiasis, including season, rainfall, and mean monthly temperature, were investigated by visual inspection of crocodile skins. Stepwise logistic regression revealed a significant negative linear relationship (P = 0.011, R (2) = 32.69 %) between mean monthly rainfall and the incidence of monthly Paratrichosoma-associated helminthiasis. Variation in the severity of Paratrichosoma-associated helminthiasis could not be explained by any of the independent environmental variables included within an ordinal regression analysis. The large genetic diversity in these nematodes indicates a high probability of anthelmintic resistant alleles occurring in the population. We discuss how the spread of these alleles may be mitigated by adopting targeted treatment protocols.

Pathology of runting in farmed saltwater crocodiles (Crocodylus porosus) in Australia.

Extremely poor growth of some individuals within a birth cohort (runting) is a significant problem in crocodile farming. We conducted a pathological investigation to determine if infectious disease is associated with runting in farmed saltwater crocodiles (Crocodylus porosus) and to look for evidence of other etiologies. In each of 2005 and 2007, 10 normal and 10 runt crocodiles, with an average age of 5.5 months and reared under identical conditions, were sampled. Laboratory testing included postmortem; histological examination of a wide variety of tissues (with quantitation of features that were noted subjectively to be different between groups); hematology; serum biochemistry (total protein, albumin, globulins, total calcium, phosphorus, and iron); bacterial culture of liver and spleen (2005 only); viral culture of liver, thymus, tonsil, and spleen using primary crocodile cell lines (2007 only); and serum corticosterone (2007 only). The only evidence of infectious disease was mild cutaneous poxvirus infection in 45% of normal and 40% of runt crocodiles and rare intestinal coccidia in 5% of normal and 15% of runt crocodiles. Bacterial and viral culture did not reveal significant differences between the 2 groups. However, runt crocodiles exhibited significant (P < .05) increases in adrenocortical cell cytoplasmic vacuolation and serum corticosterone, decreased production of bone (osteoporosis), and reduced lymphoid populations in the spleen, tonsil, and thymus. Runts also exhibited moderate anemia, hypoalbuminemia, and mild hypophosphatemia. Taken together, these findings suggest an association between runting and a chronic stress response (hyperactivity of the hypothalamic-pituitary-adrenal axis).

QTL mapping for two commercial traits in farmed saltwater crocodiles (Crocodylus porosus).

The recent generation of a genetic linkage map for the saltwater crocodile (Crocodylus porosus) has now made it possible to carry out the systematic searches necessary for the identification of quantitative trait loci (QTL) affecting traits of economic, as well as evolutionary, importance in crocodilians. In this study, we conducted genome-wide scans for two commercially important traits, inventory head length (which is highly correlated with growth rate) and number of scale rows (SR, a skin quality trait), for the existence of QTL in a commercial population of saltwater crocodiles at Darwin Crocodile Farm, Northern Territory, Australia. To account for the uncommonly large difference in sex-specific recombination rates apparent in the saltwater crocodile, a duel mapping strategy was employed. This strategy employed a sib-pair analysis to take advantage of our full-sib pedigree structure, together with a half-sib analysis to account for, and take advantage of, the large difference in sex-specific recombination frequencies. Using these approaches, two putative QTL regions were identified for SR on linkage group 1 (LG1) at 36 cM, and on LG12 at 0 cM. The QTL identified in this investigation represent the first for a crocodilian and indeed for any non-avian member of the Class Reptilia. Mapping of QTL is an important first step towards the identification of genes and causal mutations for commercially important traits and the development of selection tools for implementation in crocodile breeding programmes for the industry.

Standardized reference ideogram for physical mapping in the saltwater crocodile (Crocodylus porosus).

Basic cytogenetic data, such as diploid number and general chromosome morphology, are available for many reptilian species. Here we present a detailed cytogenetic examination of the saltwater crocodile (Crocodylus porosus) karyotype, including the creation of the first fully annotated G-band standard ideogram for any crocodilian species. The C. porosus karyotype contains macrochromosomes and has a diploid number of 34. This study presents a detailed description of each chromosome, permitting unambiguous chromosome identification. The fully annotated standardized C. porosus ideogram provides the backbone to a standard nomenclature system which can be used to accurately identify specific band locations. Seven microsatellite containing fosmid clones were fluorescently labeled and used as fluorescent in situ hybridization (FISH) probes for physical localization. Chromosome locations for each of these FISH probes were successfully assigned, demonstrating the utility of the fully annotated ideogram for genome mapping.

Quantitative analysis of production traits in saltwater crocodiles (Crocodylus porosus): III. juvenile survival.

Mortality records of 1302 juvenile crocodiles were available for analysis. Crocodiles that were slaughtered during this study were treated as censored (n = 2151). Additionally, records from animals that had neither died nor been slaughtered, i.e. were still alive in the production system (n = 1582), were censored at the last date of data collection. There were a total of 3733 censored records. The data were all full-sib records from 29 parental pairs from Janamba Croc Farm (Northern Territory, Australia), collected over nine consecutive years. Data were analysed using an extension of Cox's proportional hazards model to include frailty (random) terms to account for genetic effects. Heritability of log survival time for juvenile crocodile survival was 0.15 (SE 0.04). The probability of a juvenile crocodile surviving to day 400 was estimated to be only 51%. These results are the first to quantify juvenile survival in a captive breeding situation. Also, this is the first heritability estimate of crocodile survival and is a fundamental element in the development of a genetic improvement programme.

Quantitative analysis of production traits in saltwater crocodiles (Crocodylus porosus): IV. number of scale rows.

A total of 3156 scale row records, comprising 1739 full-sibling records from 30 families from Janamba Croc Farm (NT, Australia) and 1417 parent-offspring records from 19 families from Wildlife Management International, Pty Ltd (NT, Australia), collected at each facility using a different method, were analysed using ASReml. The full-sibling heritability estimate for the Janamba data was 0.37 (SE 0.03). The animal model estimate of heritability for the Wildlife Management International (WMI) data, also based predominantly on full-sibling data, was 0.42 (SE 0.04). The counts from three counting methods were evaluated by regression analysis on 100 individuals and were found to be highly correlated. Using the regression relationship, the WMI data were transformed and pooled with the Janamba data to give an animal model heritability estimate of 0.42 (SE 0.04). A multitrait analysis revealed negligible correlations (both phenotypical and genetical) between hatchling size traits and the number of scale rows. There is ample genetic variation to incorporate this trait into a genetic improvement programme for farmed saltwater crocodiles.

Quantitative analysis of production traits in saltwater crocodiles (Crocodylus porosus): II. age at slaughter.

Crocodile morphometric (head, snout-vent and total length) measurements were recorded at three stages during the production chain: hatching, inventory [average age (+/-SE) is 265.1 +/- 0.4 days] and slaughter (average age is 1037.8 +/- 0.4 days). Crocodile skins are used for the manufacture of exclusive leather products, with the most common-sized skin sold having 35-45 cm in belly width. One of the breeding objectives for inclusion into a multitrait genetic improvement programme for saltwater crocodiles is the time taken for a juvenile to reach this size or age at slaughter. A multivariate restricted maximum likelihood analysis provided (co)variance components for estimating the first published genetic parameter estimates for these traits. Heritability (+/-SE) estimates for the traits hatchling snout-vent length, inventory head length and age at slaughter were 0.60 (0.15), 0.59 (0.12) and 0.40 (0.10) respectively. There were strong negative genetic (-0.81 +/- 0.08) and phenotypic (-0.82 +/- 0.02) correlations between age at slaughter and inventory head length.

Quantitative analysis of production traits in saltwater crocodiles (Crocodylus porosus): I. reproduction traits.

Repeatability and phenotypic correlations were estimated for saltwater crocodile reproductive traits. No pedigree information was available to estimate heritability or genetic correlations, because the majority of breeder animals on farms were wild-caught. Moreover, as the age of the female breeders could not be accounted for, egg-size measurements were used as proxies. The reproductive traits investigated were clutch size (total number of eggs laid), number of viable eggs, number of eggs that produced a live, healthy hatchling, hatchability, average snout-vent length of the hatchlings and time of nesting. A second data set was also created comprising binary data of whether or not the female nested. Repeatability estimates ranged from 0.24 to 0.68 for the measurable traits, with phenotypic correlations ranging from -0.15 to 0.86. Repeatability for whether a female nested or not was 0.58 on the underlying scale. Correlations could not be estimated between the measurement and binary traits because of confounding. These estimates are the first published for crocodilian reproduction traits.

Analysis of microsatellites and parentage testing in saltwater crocodiles.

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.