A longitudinal study of faecal shedding of Yersinia enterocolitica and Yersinia pseudotuberculosis by Merino lambs in south-eastern Australia.

A prospective longitudinal study was conducted to investigate potential risk factors for faecal shedding of Yersinia enterocolitica and Y. pseudotuberculosis by Merino lambs in four flocks in south-eastern Australia. The primary aims of the study were to determine the seasonal patterns of shedding of pathogenic Y. enterocolitica and Y. pseudotuberculosis, and to evaluate putative risk factors for faecal shedding of these organisms, including worm egg count, live-weight and growth rate. The risk of shedding varied markedly between Yersinia spp., farms, seasons and years. Shedding of Y. pseudotuberculosis occurred predominately in winter, whereas Y. enterocolitica was commonly isolated from faeces throughout the year. Moderate to high prevalences of shedding of each organism occurred in the absence of outbreaks of yersiniosis. In general, for shedding of Y. pseudotuberculosis, animals with moderate or high worm egg counts were at increased risk of shedding compared with animals with low worm egg counts. Sheep with higher average daily weight gains were at decreased risk of shedding Y. enterocolitica but at increased risk of shedding Y. pseudotuberculosis. Live-weight was not significantly associated with risk of shedding either species. This study highlighted that exposure to determinants of shedding Y. enterocolitica and Y. pseudotuberculosis differ between farms and over time within farms. Shedding is likely influenced by environmental, animal and management factors. Our results indicate that different or additional risk factors are required for yersiniosis over those that cause faecal shedding of Yersinia spp., because moderate to high prevalences of shedding were not always associated with outbreaks of clinical disease.

Breech-strike on mulesed, clipped and unmulesed Merino ewes and hoggets in south-eastern Australia.

OBJECTIVE: Compare breech-strike on Merino ewes and hoggets that were mulesed, had breech and tail clips applied ('clipped') or left unmulesed and treated strategically with long-acting insecticide. DESIGN: A cohort study on two farms in southern Victoria. METHODS: Three treatment groups were established at lamb marking in 2008 and 2009 on each farm. The unmulesed group was treated with a long-acting insecticide in early spring. The prevalence of breech-strike and key risk factors, including presence of dag, urine stain and breech wrinkle, were compared between groups. RESULTS: Breech-strike was detected from October to December on 1.9% and 7.2% of mulesed, and on 14.8% and 12.5% of clipped ewes, respectively, on each farm. Thus, clipped ewes had a relative risk of breech-strike 7.8- and 1.7-fold that of mulesed ewes. Unmulesed ewes treated with insecticide had similar or less breech-strike compared with mulesed ewes (3.4% and 1.4%), but significantly more dag, stain and breech wrinkle. From January, breech-strike on unprotected unmulesed ewes was 8.5% and 2.8%, compared with 3.5% and 0% on mulesed ewes. CONCLUSION: Early-season treatment of unmulesed sheep prevented most breech-strikes during spring and early summer on both hoggets and breeding ewes, confirming it as a short- to medium-term option for the control of breech-strike. Some benefits were associated with the use of clips but, to control breech-strike, clipped sheep should be treated the same as unmulesed sheep. Genetic selection to reduce the prevalence of dag will be required for future strategies to control breech-strike on Merino sheep in south-eastern Australia.

Prevalence of breech-strike in mulesed, clipped and unmulesed Merino hoggets in south-eastern Australia.

OBJECTIVE: To compare the prevalence of breech flystrike ('breech-strike') on 12-20-month-old Merino hoggets that were mulesed, had breech and tail clips applied ('clipped') or were left unmulesed but treated strategically with long-acting insecticide. DESIGN: A cohort study on three farms in southern Victoria. METHODS: Three treatment groups were established at lamb marking on each farm. The unmulesed group was treated with dicyclanil when 12 months old in September or October ('early-season treatment'). The prevalence of breech-strike and key risk factors, including soiling of the breech with faeces ('dag') and urine ('stain'), and breech wrinkle, were compared between the groups. RESULTS: Over all the farms, breech-strike occurred on 1.1% of mulesed sheep and 7.7% of clipped sheep. Clipped sheep had a relative risk of breech-strike of 18.2-, 16.8- and 3.0-fold that of mulesed sheep on the three farms. Unmulesed sheep treated with dicyclanil had a similar prevalence of breech-strike to that of mulesed sheep (overall 0.9%, range 0-2.5%), but had significantly more dag, stain and wrinkle. CONCLUSION: Early-season treatment of unmulesed sheep prevented most breech-strikes during the spring and early summer, and offers a short- to medium-term option for the control of breech-strike in south-eastern Australia. There were some benefits associated with using clips, namely less dag and stain, and a shorter time spent crutching. However, to control breech-strike the clipped sheep should be treated the same as unmulesed sheep. Reducing the prevalence and severity of dag will be a major component of strategies to control breech-strike of Merinos in this region.

Financial effect of bovine Johne's disease in beef cattle herds in Australia.

OBJECTIVE: To assess the financial effect of programs for controlling bovine Johne's disease (BJD) in beef herds. DESIGN: A spreadsheet simulation model of a self-replacing beef herd in south-eastern Australia selling 400-kg steers at 15 months old. METHODS: The model calculated the monthly cash flow, and net present value (NPV) of cumulative cash flow, over 10 years. Four main control options were compared: (1) a base herd (no action to control the disease), (2) test and cull, and (3) partial and (4) total destocking. It was assumed that BJD was eradicated after 3 and 5 years with total and partial destocking, respectively, and not eradicated with a test and cull program. Scenarios were compared for both commercial and stud enterprises. RESULTS: If there was no discount on the sale price of cattle in commercial herds, deaths from BJD had to exceed 5% before the NPV of partial or total destocking was similar to taking no action to control the disease over a 10-year period. When cattle sales incurred a 10% discount, deaths had to exceed 1% before the destocking strategies would break even after 10 years. CONCLUSION: Control options for BJD should be carefully planned on an individual herd basis, as significant production and financial risks accompany destocking programs. Eradication will only be more profitable in the longer term, compared with living with the disease, when discounts on the sale of stock from infected herds are high. This can occur with the selling of store cattle or breeders. In stud herds, BJD will usually cause the total failure of the business.

Epidemiology of bovine Johne's disease (BJD) in beef cattle herds in Australia.

OBJECTIVE: To describe the epidemiology of bovine Johne's disease (BJD) in beef herds in Australia. DESIGN: Retrospective survey of beef herds detected with BJD between 1991 and 2006. METHODS: Information about the management and physical characteristics of affected herds, index and clinical cases, testing and control programs, and attitudes to BJD were collated from existing data and personal interviews of herd owners or managers. Herds were excluded if they contained fewer than 30 breeding cows or operated as a dairy farm. RESULTS: Records from 109 herds demonstrated the first detected ('index') case was 3.4-fold more likely to be a beef rather than dairy breed. However, further analysis revealed association with dairy cattle was an important risk factor for introducing BJD. Index cases were most likely detected by veterinarians investigating clinical cases of scouring or ill-thrifty animals during winter, particularly bulls or aged cows. Most herds with clinical BJD had only a single case, with only one high prevalence herd detected in the survey group. Over the period of observation, test and cull programs did not eradicate BJD unless combined with culling of known high-risk animals, but removal of high-risk cattle by partial or total destocking generally restored the trading status of affected herds. CONCLUSION: Excluding cattle with dairy contact from beef herds, ensuring more effective farm biosecurity, promptly seeking veterinary advice regarding scouring cattle and sourcing replacement cattle from demonstrably low-risk herds, such as CattleMAP and 'Beef Only' herds, are simple strategies that should reduce the risk of introducing BJD infection into beef herds.

A multi-species model to assess the effect of refugia on worm control and anthelmintic resistance in sheep grazing systems.

OBJECTIVE: Develop a computer simulation model that uses daily meteorological data and farm management practices to predict populations of Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia (Ostertagia) circumcincta and the evolution of anthelmintic resistance within a sheep flock. Use the model to explore if increased refugia, provided by leaving some adult sheep untreated, would delay development of anthelmintic resistance without compromising nematode control. PROCEDURES: Compare model predictions with field observations from a breeding flock in Armidale, NSW. Simulate the impact of leaving 1-10% of adult sheep untreated in diverse sheep-grazing systems. RESULTS: Predicted populations of Tr. colubriformis and T. circumcincta were less than those observed in the field, attributed to nutritional stress experienced by the sheep during drought and not accounted for by the model. Observed variation in faecal egg counts explained by the model (R(2) ) for these species was 40-50%. The H. contortus populations and R(2) were both low. Leaving some sheep untreated worked best in situations where animals were already grazing or were moved onto pastures with low populations of infective larvae. In those cases, anthelmintic resistance was delayed and nematode control was maintained when 1-4% of adult stock remained untreated. CONCLUSIONS: In general, the model predicted that leaving more than 4% of adults untreated did not sufficiently delay the development of anthelmintic resistance to justify the increased production risk from such a strategy. The choice of a drug rotation strategy had an equal or larger effect on nematode control, and selection for resistance, than leaving 1-10% of adults untreated.

Minimising the development of anthelmintic resistance, and optimising the use of the novel anthelmintic monepantel, for the sustainable control of nematode parasites in Australian sheep grazing systems.

OBJECTIVE: To compare the risk of different treatment scenarios on selecting for anthelmintic resistance on Australian sheep farms. DESIGN: A computer simulation model predicted populations of Trichostrongylus colubriformis, Haemonchus contortus or Teladorsagia (Ostertagia) circumcincta, and the frequency of anthelmintic resistance genes. METHOD: Nematode populations and the progression of drug resistance for a variety of treatment options and management practices in sheep-rearing areas of Western Australia (WA), Victoria (VIC) and New South Wales (NSW) were simulated. A scoring system was devised to measure the success of each option in delaying resistance to each anthelmintic and in controlling nematode populations. RESULTS: The best option at all sites was combining the new anthelmintic (monepantel) with a triple mixture of benzimidazole, levamisole and abamectin (COM). The next best option was: in NSW, rotation at each treatment between monepantel, moxidectin and COM; in VIC, rotation at each treatment between monepantel and COM; and in WA, rotation at each treatment between monepantel (used in winter) and COM or moxidectin (used in summer-autumn). In WA, rapid selection for resistance occurred as a consequence of summer-autumn treatments; however, if a small percentage of adult stock were left untreated then this selection could be greatly reduced. Despite purposely assuming relatively high resistance to benzimidazole and levamisole, COM was still effective in controlling worms and delaying resistance. CONCLUSIONS: Because of cost constraints, it may not be feasible or profitable for producers to always use the combination of all drugs. However, the second- and third-best options still considerably slowed the development of anthelmintic resistance.

Risk factors for post-weaning mortality of Merino sheep in south-eastern Australia.

OBJECTIVE: To measure associations between body weight, growth rate, sex, time of shearing and post-weaning mortality of Merino sheep. DESIGN: Uni- and multivariable survival analyses of sheep mortality during the first year after weaning, using records (n = 3657) from two field experiments conducted in Western Victoria from 1996 to 2003. RESULTS: Overall mortality was 14.3% (range 4.5-26.8%) and mean maximum mortality rate was 29 deaths/1000 weaners/month. Increased mortality risk was associated with decreases in fleece-free body weight and mean weaner growth rate, particularly at low weights and growth rates. Weaners in the lightest weaning weight quintile had a hazard ratio of 3.5, compared with the middle quintile. The hazard ratio for a 2-kg decrease in weaning weight was 1.2 to 1.7 for weaners lighter than 22 kg. The hazard ratio for a reduction in mean weaner growth rate in the first 5 months after weaning of 0.25 kg/month was 1.1 to 6.8 if mean growth rate was less than 1 kg/month, but did not differ significantly from 1 at greater growth rates. The hazard ratio for wether weaners was approximately 1.5 compared with ewe weaners. The hazard ratio for weaners shorn between December and May, compared with unshorn weaners, was 1.2 to 3.5, with the greatest risk difference associated with shearing in March (45 deaths/1000 weaners/month). CONCLUSION: Improving the body weight and mean growth rate of weaner sheep is likely to reduce post-weaning mortality. Lightweight weaners in a flock should be managed separately from the main portion after weaning. In southern Australia, not shearing spring-born Merino weaners between December and May may assist in reducing overall post-weaning mortality.

First-trimester Down syndrome screening using dried blood biochemistry and nuchal translucency.

OBJECTIVE: To assess the effectiveness of free beta-hCG, pregnancy-associated plasma protein A, and nuchal translucency in a prospective first-trimester prenatal screening study for Down syndrome and trisomy 18. METHODS: Risks were calculated for Down syndrome and trisomy 18 based on maternal age and biochemistry only (n = 10,251), nuchal translucency only (n = 5809), and the combination of nuchal translucency and biochemistry (n = 5809). RESULTS: The study population included 50 Down syndrome and 20 trisomy 18 cases. Nuchal translucency measurement was done on 33 Down syndrome and 13 trisomy 18 cases. Down syndrome screening using combined biochemistry and ultrasound resulted in a false-positive rate of 4.5% (95% confidence interval [CI] 3.9%, 5.2%) and detection rate of 87.5% (95% CI 47%, 100%) in patients under age 35 years. In older patients, the false-positive rate was 14.3% (95% CI 12.7%, 15. 8%) and detection rate was 92% (95% CI 74%, 99%). For trisomy 18 screening, the false-positive rate was 0.4% (95% CI 0.24%, 0.69%) and detection rate was 100% (95% CI 40%, 100%) in younger patients, whereas in older patients the false-positive rate was 1.4% (95% CI 0. 9%, 2.0%) and detection rate was 100% (95% CI 66%, 100%). Using modeling, at a fixed 5% false-positive rate, the Down syndrome detection rate was 91%. Conversely, at a fixed 70% Down syndrome detection rate, the false-positive rate was 1.4%. CONCLUSION: First-trimester screening for Down syndrome and trisomy 18 is effective and offers substantial benefits to clinicians and patients.

Prevention of perinatal group B streptococcal infection: current controversies.

Group B streptococcus (GBS) is the most frequent cause of neonatal sepsis in the United States. The Centers for Disease Control and Prevention (CDC) issued guidelines for its prevention in 1996. This article details areas of controversy with those guidelines and offers recommendations for resolution. We recommend that a prevention policy be adopted by all hospitals. If a screening-based policy is chosen, compliance is essential. Penicillin is the antibiotic of choice for GBS prevention. Increasing resistance to clindamycin and erythromycin might eliminate them as alternative choices in patients allergic to penicillin. Group B streptococcal prophylaxis might not be necessary in women who have repeat elective cesarean delivery. In asymptomatic women, a positive urine culture for GBS should be considered clinically equivalent to a positive vaginal or rectal sample for screening. Neonatal sepsis caused by organisms other than GBS must be monitored carefully by all hospitals providing obstetrics services.

The relationship between the rate of intake of trichostrongylid larvae and the occurrence of diarrhoea and breech soiling in adult merino sheep.

BACKGROUND AND OBJECTIVE: A hypersensitive inflammatory response, associated with the ingestion of trichostrongylid larvae, is a major cause of diarrhoea and soiling of the breech with faeces in adult Merino sheep ('hypersensitivity scours'). This response is characterised by an infiltration of eosinophils and altered lymphocyte populations in the gut of affected sheep. The objective of this study was to investigate the numbers of Ostertagia circumcincta and Trichostrongylus vitrinus infective larvae that were needed to induce diarrhoea in adult Merino sheep grazing improved pastures. METHOD: Sheep from three farms were categorised as either being susceptible, or not susceptible, to hypersensitivity scours, then transported to an experimental farm and grazed on pastures with low numbers of trichostrongylid infective larvae. They were ranked by farm of origin and previous worm egg count, then systematically allocated to one of five dose-rate groups and infected with 0, 2, 4, 10 or 20 x 10(3) trichostrongylid infective larvae per week for 13 weeks. RESULTS: In sheep selected as being susceptible to hypersensitivity scours, the lowest dose rate of infective larvae (2000 per week) was as effective in inducing diarrhoea as the highest dose rate (20,000 per week). In contrast, even the highest larval dose did not induce diarrhoea in sheep selected as not susceptible to hypersensitivity scours. Sheep selected with an increased susceptibility to hypersensitivity scours also had a significantly higher amount of moisture in their faeces at all times except during summer. CONCLUSIONS: The apparent lack of a threshold dose of trichostrongylid infective larvae needed to induce hypersensitivity scours suggests that even improved worm control programs, which substantially decrease the ingestion of larvae, may not be able to reduce the prevalence of hypersensitivity scours or the severity of breech soiling. Controlled-release anthelmintic capsules will control breech soiling but are often not a cost-effective strategy. Thus, genetic selection of less susceptible sheep is probably the best long-term option for the control of hypersensitivity scours, and the assessment of faecal moisture may be a convenient marker of susceptibility to this syndrome.

Successful management of fetal cervical teratoma using the EXIT procedure.

Fetal cervical teratoma is a cause of polyhydramnios, premature labor, and newborn airway obstruction. Formation of a multispecialty team and use of the EXIT procedure is essential for survival of the neonate. Without a team, there is little hope for fetal survival; mortality will be 80-100%. Early diagnosis and planning are essential. Cervical teratomas can contribute to pulmonary insufficiency and chondromalacia because of a mass effect in utero and underdevelopment of the fetal lungs.

The pathogenesis and control of diarrhoea and breech soiling in adult Merino sheep.

Diarrhoea and soiling of the breech with faeces ('winter scours') is a serious problem in adult Merino sheep grazing improved pastures in south-eastern Australia during winter and spring. This occurs even on farms where gastro-intestinal nematodes are effectively controlled. It was shown that winter scours was associated with the ingestion of trichostrongylid larvae, and that host factors were important in determining susceptibility to this syndrome. No differences were detected in the protective immune response of affected and unaffected sheep to gut nematodes. However, affected sheep had a hypersensitive inflammatory reaction in the pylorus and upper jejunum, characterised by the infiltration of significantly more eosinophils and changed lymphocyte populations. The changes in the lymphocyte populations included a reduced number of CD8+ cells, increased CD4+:CD8+ T-cell ratio, and significantly reduced numbers of cells reacting to interferon-gamma. High doses of infective larvae (20,000/week of Ostertagia circumcincta and Trichostrongylus vitrinus) did not induce diarrhoea in sheep not susceptible to winter scours. In contrast, only low doses (2000/week) initiated scouring in sheep selected as being susceptible to winter scours. Therefore, even considerably improved worm control programmes, including the selection of sheep with increased resistance to gut nematodes, will not prevent winter scours. Rather, phenotypic culling and genetic selection, to remove sheep susceptible to the hypersensitivity inflammatory response, is proposed as the most suitable long-term control strategy.

Effectiveness of small workshops for improving farmers' knowledge about ovine footrot.

OBJECTIVE: To determine sheep farmers' attitudes to and beliefs about ovine footrot, and to improve their knowledge about the diagnosis, control and eradication of this disease. METHOD: Eighteen workshops, involving 291 farmers, were conducted across Victoria in the spring of 1996. The workshops were designed as small-group discussions with a maximum attendance of 20 farmers to encourage active participation. All participants completed questionnaires before each workshop and 12 to 40 weeks after the last workshop. RESULTS: Before the workshops the farmers had a poor understanding of the principles of diagnosis, control and eradication of footrot. For example, only 50% knew the footrot organism survived in soil for less than 7 days, over two-thirds did not know the reason for paring sheep's feet during an eradication program, and only 31% realised cattle were a potential source of footrot infection for sheep. After the workshops, understanding about footrot was significantly improved; 87% said Dichelobacter nodosus survived in soil less than 7 days, 71% knew the reason for paring sheep's feet and 64% realised that cattle were a potential source of footrot infection. As well as improved knowledge, change of attitude among farmers is fundamentally important if virulent footrot is to be successfully controlled and eradicated. The workshops successfully initiated this process; 40% of farmers thought the workshops changed their attitudes to footrot, while 37% said they gained an increased understanding of other people's opinions about the disease. CONCLUSION: Farmers' poor understanding of ovine footrot is a constraint to the programs aimed at controlling this disease. Small group workshops may be an effective way to influence farmers' attitudes and beliefs, and could facilitate the effectiveness of regulatory disease control programs.

Comparison of urinary free beta (hCG) and beta-core (hCG) in prenatal screening for chromosomal abnormalities.

To evaluate the potential utility of free beta (hCG) and beta-core (hCG) in a prenatal screening protocol for Down syndrome we analysed these markers in dried maternal urine specimens from 163 control, 13 Down syndrome and 5 trisomy 18 pregnancies from 8 to 25 weeks' gestation. All results are reported after normalization for urinary creatinine determined by modified Jaffe reagent assay. The correlation of urinary free beta (hCG) and urinary beta-core (hCG) was 0.61 in controls and 0.93 in Down syndrome. Median MoM values in Down syndrome were 2.42 for urinary free beta (hCG) and 2.40 for beta-core (hCG). In trisomy 18 the Median MoM was 0.35 and 0.34 for free beta (hCG) and beta-core (hCG), respectively. The degree of elevation observed in DS cases with urinary free beta (hCG) is consistent with previous reports. Studies of beta-core (hCG) in Down syndrome have yielded discrepant results. In this study, beta-core (hCG) in Down syndrome is lower than values observed in early reports but consistent with more recent reports.

Prenatal maternal dried blood screening with alpha-fetoprotein and free beta-human chorionic gonadotropin for open neural tube defect and Down syndrome.

OBJECTIVE: Our purpose was to evaluate second-trimester prenatal screening for open neural tube defects and Down syndrome by use of dried blood specimen collection and transport. STUDY DESIGN: A prospective study of 7497 dried blood specimens from patients <35 years old was performed. Specimens were assayed for maternal blood alpha-fetoprotein and free beta-human chorionic gonadotropin. Patient-specific risks for both disorders were calculated and used to determine whether further evaluation was indicated. The study included an evaluation of the median and SD of analyte multiple of the median levels. RESULTS: The initial positive rate for open neural tube defect was 4.4% adjusted to 2.7% after ultrasonographic revision and collection of a second sample. The initial positive rate for Down syndrome was 3.6% adjusted to 2.8% after ultrasonographic revision. All seven cases of open neural tube defect were detected within the increased risk group. Six of 8 (75%) cases of Down syndrome were detected. The median alpha-fetoprotein multiple of the median was 3.5 in open neural tube defect cases and 0.6 in Down syndrome cases. The median free beta-human chorionic gonadotropin multiple of the median was 2.4 in Down syndrome cases. The SD (log e) of alpha- fetoprotein and free beta-human chorionic gonadotropin in 5868 unaffected white patients was 0.4022 and 0.5635, respectively. CONCLUSION: Second-trimester dried blood screening for open neural tube defects and Down syndrome can achieve screening efficiency comparable to serum-based protocols with distinct advantages over the conventional method of blood collection.

First-trimester Down syndrome screening: free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A.

OBJECTIVE: Our purpose was to determine the feasibility of a first-trimester Down syndrome screening protocol including free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A. STUDY DESIGN: First-trimester maternal blood samples from 22 Down syndrome and 483 control cases were assayed for free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A by enzyme-linked immunosorbent assay procedures. False-positive and detection rates were determined on the basis of Down syndrome risks calculated from the levels of biochemical markers and maternal age. Because 11 of the 22 Down syndrome cases were from older pregnancies (> or = 35 years old), rates were recalculated with the United States age distribution of live births to get a more representative estimate of false positives and detection efficiency. RESULTS: The median free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A levels in cases of Down syndrome was 2.09 (95% confidence interval 1.69 to 2.62) and 0.405 multiples of the median (95% confidence interval 0.28 to 0.67), respectively. At a 5.0% false-positive rate, 15 (68.2%) Down syndrome cases were detected. By the use of the age distribution of live births, 63% of cases could be expected to be detected at a 5.0% false-positive rate. CONCLUSION: First-trimester free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A screening for Down syndrome can achieve detection rates as high as those associated with alpha-fetoprotein and human chorionic gonadotropin or alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol screening in the second trimester. Prospective studies are needed to further assess first-trimester screening.

Enhanced twin pregnancy detection within an open neural tube defect and Down syndrome screening protocol using free-beta hCG and AFP.

We have applied our multimarker approach of maternal serum alpha-fetoprotein (AFP) and free-beta human chorionic gonadotropin (hCG) for Down syndrome screening to multiple gestations to assess its efficacy for improved detection of twin and triplet pregnancies. This study matched 225 cases of twin pregnancy and 39 cases of triplet pregnancy each with ten singleton pregnancies based on gestational week, race, time to receive sample, time of year of sample, and geographical area. The ratios of the MOM for each group at the tenth, 50th, and 90th percentiles were compared by the Wilcoxon test. Risks for twins were calculated using Bayes' rule, the age-related incidence of twins, and the levels of AFP and free-beta hCG. The tenth, 50th and 90th percentiles of free-beta hCG MOMs in twin and triplet cases were 0.85, 1.99, and 4.51, and 1.38, 2.78, and 4.07, respectively. For AFP, the MOMs at these percentiles were 1.26, 1.91, and 2.99, and 2.02, 2.68, and 5.30, respectively. The twin and triplet distributions for each marker were statistically significantly different from the singleton distributions (P < 0.0001) and from each other (P = 0.0012). At a twin risk cut-off of 1 in 50, 77.4 per cent of all twin gestations can be detected in a second-trimester AFP and free-beta hCG screening protocol with 5.1 per cent of singleton pregnancies falsely identified as at risk for twins. Our dual marker protocol for mid-trimester pregnancy screening combining AFP and free-beta hCG can identify over 77 per cent of twin pregnancies in women less than 35 years of age. This benefit may contribute to an improved outcome of pregnancy by early detection of multiple gestation.