Initial Case Reports of Cancer in Naked Mole-rats (Heterocephalus glaber).

Naked mole-rats (NMRs;Heterocephalus glaber) are highly adapted, eusocial rodents renowned for their extreme longevity and resistance to cancer. Because cancer has not been formally described in this species, NMRs have been increasingly utilized as an animal model in aging and cancer research. We previously reported the occurrence of several age-related diseases, including putative pre-neoplastic lesions, in zoo-housed NMR colonies. Here, we report for the first time 2 cases of cancer in zoo-housed NMRs. In Case No. 1, we observed a subcutaneous mass in the axillary region of a 22-year-old male NMR, with histologic, immunohistochemical (pancytokeratin positive, rare p63 immunolabeling, and smooth muscle actin negative), and ultrastructural characteristics of an adenocarcinoma possibly of mammary or salivary origin. In Case No. 2, we observed a densely cellular, poorly demarcated gastric mass of polygonal cells arranged in nests with positive immunolabeling for synaptophysin and chromogranin indicative of a neuroendocrine carcinoma in an approximately 20-year-old male NMR. We also include a brief discussion of other proliferative growths and pre-cancerous lesions diagnosed in 1 zoo colony. Although these case reports do not alter the longstanding observation of cancer resistance, they do raise questions about the scope of cancer resistance and the interpretation of biomedical studies in this model. These reports also highlight the benefit of long-term disease investigations in zoo-housed populations to better understand naturally occurring disease processes in species used as models in biomedical research.

Renal Pathology in a Nontraditional Aging Model: The Naked Mole-Rat (Heterocephalus glaber).

The naked mole-rat (NMR; Heterocephalus glaber) is growing in popularity as a model for aging research due to its extreme longevity (up to 30 years), highly adapted physiology, and resistance to cancer, particularly when compared with traditional aging models such as laboratory mice and rats. Despite the NMR's seemingly lengthy health span, several age-related lesions have been documented. During a 15-year retrospective evaluation of a zoo-housed population, histologic changes in the kidneys were reported in 127 of 138 (92%) adult NMRs. Of these, renal tubular mineralization was very common (115 of 127; 90.6%) and found in NMRs without concurrent renal lesions (36 of 127; 28.3%). Many of the other described lesions were considered progressive stages of a single process, generally referred to as chronic nephritis or nephropathy, and diagnosed in 73 of 127 (57.5%), while end-stage renal disease was reported in only 12 (9.4%) NMRs. Renal lesions of these NMRs were comparable to disease entities reported in laboratory rats and certain strains of inbred and noninbred mice. Although many lesions of NMR kidneys were similar to those found in aged laboratory rodents, some common urinary diseases were not represented in the examined colonies. The goal of this study was to describe renal lesions in NMRs from a zoologic setting to familiarize investigators and pathologists with an apparently common and presumably age-related disease in this nontraditional model.

Spontaneous histologic lesions of the adult naked mole rat (Heterocephalus glaber): a retrospective survey of lesions in a zoo population.

Naked mole rats (NMRs; Heterocephalus glaber) are highly adapted, subterranean, eusocial rodents from semiarid regions of the eastern horn of Africa and the longest-living rodent known with a maximum life span of up to 30 years. They are a unique model for aging research due to their physiology, extreme longevity, and, when compared to mice and rats, resistance to cancer. Published surveys of disease in NMRs are sparse. Captive colonies in zoological collections provide an opportunity to monitor spontaneous disease over time in a seminatural environment. This retrospective study describes common lesions of a zoo population over a 15-year period during which 138 adult NMRs were submitted for gross and histologic evaluation. Of these, 61 (44.2%) were male, 77 (55.8%) female, 45 (32.6%) died, and 93 (67.4%) were euthanized. The most frequent cause of death or reason for euthanasia was conspecific trauma (bite wounds) and secondary complications. Some common histologic lesions and their prevalence were renal tubular mineralization (82.6%), hepatic hemosiderosis (64.5%), bite wounds (63.8%), chronic progressive nephropathy (52.9%), and calcinosis cutis (10.1%). In sum, 104 (75.4%) NMRs had more than one of the most prevalent histologic lesions. No malignant neoplasms were noted; however, there was a case of renal tubular adenomatous hyperplasia with nuclear atypia and compression that in rats is considered a preneoplastic lesion. This retrospective study confirms the NMR's relative resistance to cancer in spite of development of other degenerative diseases and highlights the utility of zoological databases for baseline pathological data on nontraditional animal models.

Occlusive fungal tracheitis in 4 captive bottlenose dolphins (Tursiops truncatus).

Respiratory disease is common in dolphins, primarily affecting pulmonary parenchyma and sparing large airways. Over a 10-year period, 4 captive adult bottlenose dolphins succumbed to chronic, progressive respiratory disease with atypical recurrent upper respiratory signs. All dolphins had severe, segmental to circumferential fibrosing tracheitis that decreased luminal diameter. Histologically, tracheal cartilage, submucosa, and mucosa were distorted and replaced by extensive fibrosis and pyogranulomatous inflammation centered on fungal hyphae. In 3 of 4 cases, hyphae were morphologically compatible with Aspergillus spp and confirmed by culture in 2 cases. Amplification of fungal DNA from tracheal tissue was successful in one case, and sequences had approximately 98% homology to Aspergillus fumigatus. The remaining case had fungi compatible with zygomycetes; however, culture and polymerase chain reaction were unsuccessful. Lesions were evaluated immunohistochemically using antibodies specific to Aspergillus spp. Aspergillus-like hyphae labeled positively, while presumed zygomycetes did not. These cases represent a novel manifestation of respiratory mycoses in bottlenose dolphins.

Neuronal embryonal tumors in fish.

This article describes 11 cases of neuronal embryonal neoplasia in captive adult teleost fish. Neoplasms were located within 1 or both eyes of 8 fish and the skin of 3 other fish. Ocular neoplasms most often presented as unilateral or bilateral exophthalmia. Seven ocular and 1 cutaneous mass were composed of small triangular (carrot-shaped) neoplastic cells with Flexner-Wintersteiner-type rosette formation. Mass location and histologic and ultrastructural features were suggestive of retinoblastomas. One ocular mass was composed of ribbons and rosettes of neoplastic cells with multiple areas of neuronal differentiation and was diagnosed as a teratoid medulloepithelioma. A cutaneous mass from an electric eel (Electrophorus electricus) consisted of rosettes and streams of elongate neoplastic cells. The epidermal electroreceptor (ampullary) organ was considered as an origin. Although distant metastases were not observed, neoplasms were generally locally aggressive with postexcision recurrence. There was occasional spread to or de novo occurrence within the contralateral eye.

Morphologic evidence suggestive of hypertension in western gray kangaroos (Macropus fuliginosus).

Marked renal vascular changes, suggestive of hypertension, were present in adult western gray kangaroos (Macropus fuliginosus) from a single facility over a 14-year period. A subset of these kangaroos also had vague clinical nervous system deficits, including blindness. To characterize the vascular lesions, determine prevalence, and document other changes, case histories and archival tissue sections from 21 adult kangaroos (8 male, 13 female) that died or were euthanatized between 1994 and 2008 were reviewed. Relevant lesions included increased thickness of the renal arteriolar tunica media with smooth muscle hypertrophy and/or hyperplasia, accumulation of extracellular matrix within arterioles, increased vascular tortuosity, and varying degrees of juxtaglomerular hyperplasia. Renal tissue from two more severely affected animals was further examined by transmission electron microscopy, highlighting arteriolar endothelial cell hypertrophy and disruption of the medial architecture. Hypertrophy of arteries and arterioles in other organ systems was also present (3/21), including vessels in the brain and spinal cord of one animal with clinical neurologic signs. Four kangaroos had antemortem retinal detachment, a potential sequel of hypertension in humans and domestic mammals. The cause of these vascular lesions in this mob is uncertain. Lesions were not associated with an infectious disease process, age, underlying renal disease, or thyroid abnormalities. In the absence of other causes, hypertension was a differential. Further investigation into clinical significance and predisposing factors, such as genetics and diet, is warranted.

The 2008 ACVP role delineation survey and initial data analysis: from the Role Delineation Task Force.

The American College of Veterinary Pathologists commissioned a role delineation survey to define the specialized tasks, knowledge, and tools that define the current practice of veterinary clinical pathology and veterinary anatomic pathology. The survey also identified when competence was acquired for each task (i.e., before certification or after certification). The response rate by diplomates was high, with approximately 50% of practicing pathologists within each specialty responding to each survey. Using the survey results, all tasks for each specialty were classified as either appropriate or unsuitable for testing in the certifying examinations. The role delineation survey data will facilitate the creation of test plans that objectively define the content in each certifying examination, the evaluation and enhancement of training curricula, and the optimization of continuing education opportunities for practicing veterinary pathologists.

Prevalence and pathology of lungworm infection in bottlenose dolphins Tursiops truncatus from southwest Florida.

Parasitism of the respiratory system is a relatively common finding in stranded cetaceans; however, no systematic investigations regarding the severity, distribution, and clinical consequences of these infections in bottlenose dolphins Tursiops truncatus have been conducted previously. The present study determined the prevalence of lungworm infections in dead stranded (n=22) and live bottlenose dolphins (n=44) from southwestern Florida, USA, during the period from 2003 to 2005. Dead stranded bottlenose dolphins were necropsied and lungs were examined visually, by palpation, and histologically for lesions consistent with verminous pneumonia. When present, nematodes were counted, measured, and identified to species based upon their morphology. Dolphin feces and blowhole swabs were collected and examined for nematode larvae. Lungworm prevalence was 77% in dead animals (n=22). The lesions in most cases were mild, chronic, and not the primary cause of death. Only 13% of dead animals examined had patent infections, with larvae present in blowhole and fecal cytology, and only 18% of animals had intact worms present at necropsy, with a geometric mean intensity of infection of 22.6 worms animal(-1). Intact worms were identified as either Halocercus lagenorhynchi or Skrjabinalius cryptocephalus. The highest prevalence of active infections was found in neonates and calves, including 1 stillborn calf. For free-ranging animals, all blowhole swabs (n=44) were negative, and fecal cytology (n=22) showed a 3% prevalence of patent infection. Findings from the present study support the theory that bottlenose dolphins can be infected transplacentally by lungworms. The impact that such infections may have on neonatal survival is unknown; however, these infections could increase neonatal mortality.

Predicting somatic cell count standard violations based on herd's bulk tank somatic cell count. Part I: Analyzing variation.

The present study examines the relationship between the bulk tank somatic cell count (SCC) mean and sigma (an estimate of variation) and the probability of exceeding a SCC standard. Daily or every other day, bulk tank SCC data were collected for 24 mo from 1,501 herds. Mean and sigma were estimated for each herd monthly and were compared between months and herd production categories using Kruskal-Wallis nonparametric ANOVA. The effect of month on bulk tank SCC mean and sigma was significant, with estimates for all summer months and most of the spring and fall months being significantly greater than estimates of mean and sigma in December 2004. Logistic regression models were developed to examine the relationship between month and herd production and the odds of a herd exceeding a SCC standard. The odds of exceeding a bulk tank SCC standard were significantly greater in the summer months and for smaller herds. A grid was constructed determining the probability of exceeding any of 5 SCC standards (200,000 to 600,000 cells/mL, step 100,000 cells/mL) in the following month, based on the mean and sigma of the past month. The violation probability grid can be used to assess the prospect of meeting quality premium goals and to proactively encourage more consistent performance in all the processes affecting bulk tank SCC.

Predicting somatic cell count standard violations based on herd's bulk tank somatic cell count. Part II: Consistency index.

The present study examines the capability of 1,501 herds in the Upper Midwest and the performance of statistical process control charts and indices as a way of monitoring and controlling milk quality on the farm. For 24 mo, daily or every other day bulk tank somatic cell count (SCC) data were collected. Consistency indices for 5 different SCC standards were developed. The indices calculate the maximum variation allowed to meet a desired SCC level at a given mean bulk tank SCC and were used to identify herds not capable of meeting a specific SCC standard. Consistency index method was compared with a test identifying future bulk tank SCC standard violators based on herds' past violations. The performance of the consistency index test and the past violation method was evaluated by logistic regression. The comparison focused on detection probability and certainty associated with a result. For the 5 SCC levels, detection probability and certainty associated with a result ranged from 51 to 98%. Detection probability of all violators and certainty associated with a negative result was greater for the consistency index across all 5 SCC levels (by 0.7 to 7.4% and 2.1 to 5.1%, respectively). Control charts were plotted and monthly consistency indices calculated for individual farms. Charts in combination with the consistency indices would warn from 66 to 80% of the herds about an upcoming violation within 30 d before it occurred. They offer a proactive approach to maintaining consistently high milk quality. By assessing process capability and distinguishing between significant changes and random variation in bulk tank SCC, tools presented in this article encourage fact-based decisions in dairy farm milk quality management.

Early manifestations and spectrum of recipient twin cardiomyopathy in twin-twin transfusion syndrome: relation to Quintero stage.

OBJECTIVES: To examine cardiac structural and functional changes in twin-twin transfusion syndrome (TTTS), relative to Quintero stage, as a means of evaluating the spectrum of cardiomyopathy in TTTS. METHODS: This was a cross-sectional, retrospective study of 42 consecutive cases of TTTS referred to a single fetal therapy center. Quintero stages were assigned by standard criteria. Presence of ventricular hypertrophy, cardiomegaly, atrioventricular valve regurgitation (AVVR), ventricular systolic dysfunction and right ventricular outflow tract obstruction on fetal echocardiography were noted. The Doppler myocardial performance index (MPI), an index of global ventricular function, was calculated for both ventricles in subjects with adequate Doppler data. We compared cardiac changes across Quintero stages. RESULTS: There was no cardiomyopathy observed in donor twins. The majority of subjects presented at Quintero Stage I (n = 14), II (n = 14) or III (n = 11), with fewer at Stages IV (n = 2) or V (n = 1). As early as Quintero Stages I and II, a significant proportion of recipient twins had ventricular hypertrophy (17/28, 61%), AVVR (6/28, 21%) or quantitative abnormalities in either right (12/24, 50%) or left (14/24, 58%) ventricular function. Increasing prevalence of biventricular systolic dysfunction and cardiomegaly accompanied advancing Quintero stage. CONCLUSIONS: Changes in cardiac structure and function not reflected in Quintero staging occur in recipient twins early in the evolution of TTTS. Incorporation of cardiac findings into assessment of TTTS severity may prove useful in stratification of risk and treatment selection.

Bulk tank somatic cell counts analyzed by statistical process control tools to identify and monitor subclinical mastitis incidence.

The objective of this study was to examine the relationship between monthly Dairy Herd Improvement (DHI) subclinical mastitis and new infection rate estimates and daily bulk tank somatic cell count (SCC) summarized by statistical process control tools. Dairy Herd Improvement Association test-day subclinical mastitis and new infection rate estimates along with daily or every other day bulk tank SCC data were collected for 12 mo of 2003 from 275 Upper Midwest dairy herds. Herds were divided into 5 herd production categories. A linear score [LNS = ln(BTSCC/100,000)/0.693147 + 3] was calculated for each individual bulk tank SCC. For both the raw SCC and the transformed data, the mean and sigma were calculated using the statistical quality control individual measurement and moving range chart procedure of Statistical Analysis System. One hundred eighty-three herds of the 275 herds from the study data set were then randomly selected and the raw (method 1) and transformed (method 2) bulk tank SCC mean and sigma were used to develop models for predicting subclinical mastitis and new infection rate estimates. Herd production category was also included in all models as 5 dummy variables. Models were validated by calculating estimates of subclinical mastitis and new infection rates for the remaining 92 herds and plotting them against observed values of each of the dependents. Only herd production category and bulk tank SCC mean were significant and remained in the final models. High R2 values (0.83 and 0.81 for methods 1 and 2, respectively) indicated a strong correlation between the bulk tank SCC and herd's subclinical mastitis prevalence. The standard errors of the estimate were 4.02 and 4.28% for methods 1 and 2, respectively, and decreased with increasing herd production. As a case study, Shewhart Individual Measurement Charts were plotted from the bulk tank SCC to identify shifts in mastitis incidence. Four of 5 charts examined signaled a change in bulk tank SCC before the DHI test day identified the change in subclinical mastitis prevalence. It can be concluded that applying statistical process control tools to daily bulk tank SCC can be used to estimate subclinical mastitis prevalence in the herd and observe for change in the subclinical mastitis status. Single DHI test day estimates of new infection rate were insufficient to accurately describe its dynamics.

Neospora caninum infection in a free-ranging raccoon (Procyon lotor) with concurrent canine distemper virus infection.

During a canine distemper virus (CDV) outbreak in raccoons (Procyon lotor) from Cook County, Illinois, a juvenile female suffering from seizures was killed and necropsied. Gross and histologic findings of necrotizing encephalitis and proliferative bronchopneumonia were attributed to CDV infection and considered the cause of clinical signs. A section of cerebellum stained immunohistochemically for Neospora caninum revealed an approximately 40 microm diameter, round to oval cyst with a 2- to 3-microm-thick wall and filled with 1-2 microm diameter, round to oval bradyzoites. Polymerase chain reaction (PCR) results were positive for N. caninum using DNA extracted from the brain. Specific PCR for the closely related organisms Toxoplasma gondii and Hammondia heydorni yielded negative results. This case report provides histologic, immunohistochemical, and molecular evidence that raccoons are a naturally occurring intermediate host of N. caninum.

Helicobacter cetorum sp. nov., a urease-positive Helicobacter species isolated from dolphins and whales.

A novel helicobacter with the proposed name Helicobacter cetorum, sp. nov. (type strain MIT 99-5656; GenBank accession number AF 292378), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins (Lagenorhynchus acutus) and from the feces of three captive cetaceans (a Pacific white-sided dolphin [Lagenorhynchus obliquidens]; an Atlantic bottlenose dolphin [Tursiops truncatus]; and a beluga whale [Delphinapterus leucas]). The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy. Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals. In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria. Helicobacter cetorum is a fusiform gram-negative bacterium with a single bipolar flagellum. The isolates grow under microaerobic conditions at 37 and 42 degrees C but not at 25 degrees C. H. cetorum is urease, catalase, and oxidase positive, and it is sensitive to cephalothin. The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant. By 16S rRNA sequencing it was determined that H. cetorum represented a distinct taxon that clusters most closely with H. pylori. Further studies are necessary to determine the role of H. cetorum in the development of gastric ulcers and gastritis of cetaceans. This is the first description and formal naming of a novel Helicobacter species from a marine mammal.

Characterizing biosecurity, health, and culling during dairy herd expansions.

Our objectives were to investigate strategies for biosecurity, expansion, and culling for expanding dairy herds in the Upper Midwest. Eighteen dairies in Iowa and Wisconsin were visited, and dairy managers and veterinarians were interviewed to characterize five biosecurity practices, herd culling practices, vaccines administered, and ensuing disease status for the herds. The majority of herds that were interviewed failed to employ comprehensive biosecurity programs for incoming cattle. Nearly 60% of herds obtained cattle from sources for which it was difficult to document genetic backgrounds and health histories, fewer than half required health testing for incoming cattle, and approximately 50% quarantined new cattle on arrival. Despite high rates of vaccination for bovine viral diarrhea, all herd owners and managers indicated that herd biosecurity was compromised as a result of expansion. Half of the interviewed herds indicated that bovine viral diarrhea and papillomatous digital dermatitis were notable disease problems. Herds that obtained cattle with unknown backgrounds and health status experienced the largest number of diseases. Before expansion, the most frequently cited reasons for culling were reproductively unsound; low milk production; mastitis, poor udder health, and high SCC; during expansion, the strategic decision to cull cows for low milk production was used less often. In addition, the stochastic simulation model, DairyORACLE, was used to evaluate economic outcomes for several expansion alternatives. Five model scenarios studied were: base scenario (herd size was maintained) and four expansion scenarios--all paired combinations of heifer quality (high, low) and voluntary culling (implemented, not implemented). Culling for low milk production yielded an additional $23.29 annually (6-yr annuity) per cow, but on the basis of purchased replacements, no voluntary culling was most profitable. Purchasing high versus low quality replacement heifers for expansions returned an additional $113.54 annually ($681.24 total net present value) per heifer purchased. Many opportunities exist to improve cattle-related factors for dairy herd expansions, including the use of comprehensive biosecurity programs, realistic planning and budgeting for cattle purchases, and cost effective purchase and culling practices.

A new species of Eimeria (Apicomplexa, Eimeriidae) from the weedy sea dragon Phyllopteryx taeniolatus (Osteichthyes: Syngnathidae).

A new species of intestinal coccidian is described from the weedy or common sea dragon Phyllopteryx taeniolatus housed at the New England Aquarium in Boston and at the Shedd Aquarium in Chicago, USA. Live oocysts of Eimeria phyllopterycis sp. n. are spherical, 30.9 (28.0-34.4) microm, with a thin, single-layered wall. Both a micropyle and oocyst residuum are absent and a large polar granule is sometimes present. Sporocysts are ellipsoidal and elongate, 24.3 x 10.4 (23.4-25.6 x 9.2-11.2) microm, with Stieda and substieda bodies; shape index (length/width) 2.33 (2.14-2.70). A sporocyst residuum is present, consisting of numerous granules of various sizes. Sporozoites each possess 3 refractile bodies. Preliminary evidence suggests that the coccidian may affect the health of sea dragons; however, it could not be determined whether this parasite caused significant morbidity or mortality.

Conformations of protonated gas-phase bradykinin ions: evidence for intramolecular hydrogen bonding.

The post-source decay of bradykinin, Lys1-bradykinin, des-Arg1-bradykinin, des-Arg9-bradykinin and [D-Phe7]-bradykinin [M + H]+ ions was examined in order to assertain the influence of secondary structure on peptide ion dissociation. Fragment ions corresponding to the elimination of H2O and HN=C=NH are observed in the product ion mass spectra of Lys1-bradykinin and des-Arg1-bradykinin but not in the spectra of bradykinin or des-Arg9-bradykinin. Cleavage reactions at the Phe-Ser and/or Ser-Pro bonds are observed for all peptide [M + H]+ ions with the exception of des-Arg9-bradykinin. The product ions arising from the processes described above are rationalized in terms of the intramolecular solvation of the protonated guanidino groups of the arginines. The strongest intramolecular interaction appears to be a proton bridge between the guanidino groups of the N- and C-terminal arginines in bradykinin. In addition, increased abundances of fragment ions in the vicinity of Ser-Pro may be attributed to intramolecular solvation of the protonated C-terminal guanidino group by the Ser-Pro portion of the molecule. This self-solvation of the ionizing proton leads to a gas-phase peptide conformation that is supported by solution-phase NMR studies at elevated temperatures and in non-polar solvents but which is different from the conformation in polar solvents.

Gastric spiral bacteria in small felids.

Nine small cats, including one bobcat (Felis rufus), one Pallas cat (F. manul), one Canada lynx (F. lynx canadensis), two fishing cats (F. viverrina), two margays (F. wiedii), and two sand cats (F. margarita), necropsied between June 1995 and March 1997 had large numbers of gastric spiral bacteria, whereas five large cats, including one African lion (Panthera leo), two snow leopards (P. uncia), one Siberian tiger (P. tigris altaica), and one jaguar (P. onca), necropsied during the same period had none. All of the spiral organisms from the nine small cats were histologically and ultrastructurally similar. Histologically, the spiral bacteria were 5-14 microm long with five to nine coils per organism and were located both extracellularly within gastric glands and surface mucus, and intracellularly in parietal cells. Spiral bacteria in gastric mucosal scrapings from the Canada lynx, one fishing cat, and the two sand cats were gram negative and had corkscrewlike to tumbling motility when viewed with phase contrast microscopy. The bacteria were 0.5-0.7 microm wide, with a periodicity of 0.65-1.1 microm in all cats. Bipolar sheathed flagella were occasionally observed, and no periplasmic fibrils were seen. The bacteria were extracellular in parietal cell canaliculi and intracellular within parietal cells. Culture of mucosal scrapings from the Canada lynx and sand cats was unsuccessful. Based on morphology, motility, and cellular tropism, the bacteria were probably Helicobacter-like organisms. Although the two margays had moderate lymphoplasmacytic gastritis, the other cats lacked or had only mild gastric lymphoid infiltrates, suggesting that these organisms are either commensals or opportunistic pathogens.

Risk factors for twinning in dairy cows.

Records for 52,362 lactations over a 10-yr period from 260 dairy farms in North America that used a common commercial software for record keeping were evaluated for potential risk factors for twinning. Records were evaluated for the associations of reproductive disease, parity, production, drug therapy, and the occurrence of subsequent twins. The rate of twinning on these farms steadily increased over the observation period from 1.4% in 1983 to 2.4% in 1993. The rate of twinning also increased as parity of the cow increased, from 1.0% for cows in their first lactation to > 4.1% for cows in their fifth or higher lactation. No association between twinning and season of year was detected. A multivariate logistical regression analysis found that the rate of twinning increased with increases in milk production, incidence of cystic ovarian disease, and the use of common pharmaceuticals, including GnRH, PGF2 alpha, and antibiotics. Results of the regression model also indicated that the single most important reason for the recent increase in the rate of twinning was a concurrent increase in milk production.