Epidemiology of prolonged testicular infections with bovine viral diarrhea virus.

Previously, bovine viral diarrhea virus (BVDV) had been found in prolonged testicular infections following acute infection of immunocompetent bulls. The primary purpose of this research was to evaluate the production and maintenance of prolonged testicular infections after exposure to BVDV of seronegative bulls in varying circumstances. The secondary objective was to initiate assessment of the potential for transmission of BVDV via semen of bulls exhibiting a prolonged testicular infection. In total, 10 research trials were conducted. The first trial examined the duration of detectable virus in semen after intranasal inoculation of peri-pubertal bulls. The second to fifth trials examined the potential for prolonged testicular infections resulting from natural exposure of seronegative bulls to persistently infected heifers. In the last five trials, the potential for viral transmission from bulls exhibiting prolonged testicular infections to a small number of exposed animals (n=28) was evaluated. Results of this research demonstrated that prolonged testicular infections could result in detection of viral RNA in semen for 2.75 years with infectious virus grown from testicular tissue 12.5 months after viral exposure. A type 1b strain of BVDV caused prolonged testicular infection after natural exposure of seronegative bulls to a persistently infected heifer. However, transmission of BVDV to susceptible animals was not detected in the final five trials of this research. In conclusion, BVDV can persist in testicular tissue after acute infection for several years, but the potential for viral transmission from these prolonged testicular infections appears to be low.

Comparison of tests for detection of bovine viral diarrhea virus in diagnostic samples.

Currently, a variety of tests are used to detect bovine viral diarrhea virus (BVDV) in persistently infected (PI) cattle. These tests include immunohistochemical staining (IHC), antigen capture enzyme-linked immunosorbent assay (ACE), virus isolation (VI), and reverse transcription-polymerase chain reaction (RT-PCR). However, a lack of methods standardization could compromise the ability to consistently identify animals infected with BVDV. This study evaluated the diagnostic proficiency of current methods for detecting BVDV in infected cattle using intra- and interlaboratory comparisons. Samples were collected from 4 animals more than 7 months of age (2 BVDV negative animals, a PI animal, and a PI animal that previously lacked detectable virus in serum as determined by VI). Samples were submitted to 23 participating diagnostic laboratories using the respective laboratory's standard submission protocol. Samples collected for submission included: 1) serum for ACE, RT-PCR, and VI; 2) whole blood for RT-PCR and VI; and 3) skin biopsies for ACE and IHC. The ACE performed on skin provided the greatest consistency in detecting positive samples and a perfect level of agreement among laboratories. Reverse transcription-polymerase chain reaction and IHC performed well by correctly identifying > or = 85% of samples positive for BVDV. Virus isolation performed on serum yielded the lowest consistency in detecting positive samples and the lowest level of agreement. The level of agreement between laboratories for detecting BVDV in persistently infected cattle ranged from perfect to less than expected by chance. The variation between laboratories suggests a need for training opportunities in standardized laboratory protocols and proficiency testing.

Use of three-dimensional computed tomography for diagnosis and treatment planning for open-mouth jaw locking in a cat.

CASE DESCRIPTION: A 2.5-year-old spayed female Persian cat was evaluated for acute inability to close its mouth. CLINICAL FINDINGS: A wry-mouth malocclusion was evident, and the right side of the mandible was longer than the left side. The right mandibular tooth row appeared to be lowered. The lower jaw was persistently maintained in an open position. The presumptive diagnosis was open-mouth jaw locking. Diagnostic imaging with computed tomography and 3-dimensional reconstruction was performed for definitive diagnosis and to achieve a better understanding of the lesions. Imaging revealed locking of the right ramus of the mandible, which was displaced ventrolaterally, causing the coronoid process to impinge on the right zygomatic arch. TREATMENT AND OUTCOME: A bilateral partial ostectomy of the rostroventral margins of the zygomatic arches with an autogenous fat graft implantation was performed. The cat recovered without complications and by the following morning was bright, alert, and responsive and eating canned cat food comfortably. One year after surgery, the owner reported that the cat had continued to function well, was eating normally, and had not had any observed locking episodes since surgery. CLINICAL RELEVANCE: Unlike radiographic imaging, computed tomography may be used to create 3-dimensional reconstructions of structures in cases of suspected open-mouth jaw locking; improve evaluation of the lesions; and improve decision-making and client education for diagnosis, treatment options, and prognosis.

Noncytopathic bovine viral diarrhea virus can persist in testicular tissue after vaccination of peri-pubertal bulls but prevents subsequent infection.

The objectives of this research were to evaluate the risk of prolonged testicular infection as a consequence of vaccination of peri-pubertal bulls with a modified-live, noncytopathic strain of BVDV and to assess vaccine efficacy in preventing prolonged testicular infections after a subsequent acute infection. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with an approximate minimum immunizing dose or a 10x standard dose of modified-live, noncytopathic BVDV or were maintained as unvaccinated controls. Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry and the identity of viral strains was determined by nucleotide sequencing of PCR products. The vaccine strain of BVDV was detected in testicular tissue of vaccinated bulls as long as 134 days after immunization. Prolonged testicular infections with the challenge strain were detected only in unvaccinated bulls as long as 85 days after challenge. Whereas vaccination caused prolonged testicular infection in some bulls, it did prevent subsequent infection of testicular tissue with the challenge strain. This research demonstrates that subcutaneous vaccination of naïve, peri-pubertal bulls with a noncytopathic, modified-live strain of BVDV can result in prolonged viral replication within testicular tissue. The risk for these prolonged testicular infections to cause venereal transmission of BVDV or subfertility is likely to be low but requires further investigation.

Use of a modified-live vaccine to prevent persistent testicular infection with bovine viral diarrhea virus.

A commercial vaccine containing modified-live bovine viral diarrhea virus (BVDV; types 1 and 2) was administered to one group of 22 peripubertal bulls 28 days before intranasal inoculation with a type 1 strain of BVDV. A second group of 23 peripubertal bulls did not receive the modified-live BVDV vaccine before intranasal inoculation. Ten of 23 unvaccinated bulls--but none of the vaccinated bulls--developed a persistent testicular infection as determined by immunohistochemistry and polymerase chain reaction. Results of this study indicate that administration of a modified-live vaccine containing BVDV can prevent persistent testicular infection if peripubertal bulls are vaccinated before viral exposure.

Seroconversion of calves following intravenous injection with embryos exposed to bovine viral diarrhea virus (BVDV) in vitro.

Two recent studies demonstrated that a high-affinity isolate of BVDV (SD-1), remained associated with a small percentage of in vivo-derived bovine embryos following artificial exposure to the virus and either washing or trypsin treatment. Further, the embryo-associated virus was infective in an in vitro environment. Therefore, the objective of this study was to determine if the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos could cause infection in vivo. Twenty zona-pellucida-intact morulae and blastocysts (MB) were collected on day 7 from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 MB (negative controls) were exposed for 2 h to 10(5)-10(6) cell culture infective doses (50% endpoint) per milliliter (CCID(50)/mL) of viral strain SD-1. Following exposure, according to IETS standards, one half of the MB were washed and one half were trypsin treated. All MB were then individually sonicated, and sonicate fluids were injected intravenously into calves on day 0. Blood was drawn to monitor for viremia and(or) seroconversion. Seroconversion of calves injected with sonicate fluids from washed and trypsin-treated embryos occurred 38% and 13% of the time, respectively. Therefore, the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos was infective in vivo.

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos.

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.

Different strains of noncytopathic bovine viral diarrhea virus (BVDV) vary in their affinity for in vivo-derived bovine embryos.

Washing procedures (without trypsin treatment) recommended by the International Embryo Transfer Society (IETS) for use on in vivo-derived embryos effectively removed a cytopathic strain (NADL) of bovine viral diarrhea virus (BVDV) after artificial exposure. However, these washing procedures have not been evaluated using other isolates of BVDV, including representative non-cytopathic strains. Thus, the objective of this study was to evaluate the efficacy of the IETS procedures following artificial exposure of in vivo-derived bovine embryos to two different strains and biotypes of BVDV. One hundred and twenty-nine zona pellucida-intact (ZP-I) morulae and blastocysts (MB) and 56 non-fertile and degenerated (NFD) ova were collected 7 days following exposure to bulls from 32, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to IETS standards. Subsequently, half of the MB and NFD ova were exposed for 1h to approximately 10(6)-cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1, and the other half were exposed to the same concentration of CD-87. After exposure, groups of > or =3 and < or = 10 MB or NFD ova were washed using methods that met or exceeded IETS standards. Then, the washed groups were sonicated, and sonicate fluids were assayed for presence of virus using virus isolation and a reverse transcription nested polymerase chain reaction. No virus was detected in any group of MB or NFD ova that had been exposed to the CD-87 isolate. However, virus was detected in association with 50% of the groups of MB and 33% of the groups of NFD ova that had been exposed to the SD-1 isolate. Therefore, standard embryo-washing procedures recommended by the IETS are more effective for removal of some isolates of BVDV than for others. It remains to be determined if the quantity of a high-affinity isolate of BVDV associated with individual washed embryos would infect recipients via the intrauterine route. Further, it should be determined if an alternative embryo processing procedure, washing and trypsin treatment, would be more effective for removal of high-affinity isolates.

Detection of bovine viral diarrhea virus in semen obtained after inoculation of seronegative postpubertal bulls.

OBJECTIVE: To evaluate persistence of bovine viral diarrhea virus (BVDV) in semen after inoculation of postpubertal bulls. ANIMALS: Three 2-year-old bulls and five 6-month-old calves. PROCEDURE: 3 seronegative 2-year-old bulls were inoculated intranasally with BVDV. Serum and semen samples were obtained at regular intervals until 7 months after inoculation. Serum samples were tested for BVDV by use of virus isolation (VI) and reverse transcription-nested polymerase chain reaction (RT-nPCR) tests. Semen samples were tested for virus by use of VI and RT-nPCR tests. Testicular biopsy specimens were obtained 7 months after inoculation and tested for BVDV by use of immunohistochemical analysis and VI and RT-nPCR tests. Semen samples collected from 1 bull immediately before and 5 and 7 months after inoculation were administered IV to seronegative calves, which were monitored for subsequent viremia and seroconversion. RESULTS: Use of VI and RT-nPCR tests detected transient virus in serum of all bulls. The VI test detected BVDV in semen of 2 bulls for < 21 days after inoculation, whereas RT-nPCR assay detected BVDV until 7 months after inoculation. Virus was detected in testicular biopsy specimens of these 2 bulls by use of immunohistochemical analysis and RT-nPCR assay but could only be isolated from the biopsy specimen of 1 bull. Of the calves administered semen IV to detect infectious virus, only the recipient of semen collected 5 months after inoculation of the adult bull was viremic and seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine viral diarrhea virus can persist in semen of acutely infected bulls for several months after exposure.

Effects of testicular biopsy in clinically normal bulls.

OBJECTIVE: To determine whether testicular needle biopsy is detrimental to testicular function in clinically normal bulls. DESIGN: Prospective study. ANIMALS: 6 mixed-breed mature bulls. PROCEDURE: A randomly selected testicle from each bull was biopsied with a 14-gauge needle biopsy instrument. Bulls were then evaluated over a 90-day period for changes in scrotal temperature and thermal patterns, ultrasonographic appearance, and quality of spermatozoa. At the end of the 90-day study, bulls were castrated, and testicles were examined grossly and histologically. RESULTS: Changes were detected in scrotal temperatures and thermal patterns and in the breeding soundness examination results during the first 2 weeks of the study. However, there were no long-term changes in semen quality over the course of the experiment. Hyperechoic areas were detected on ultrasonographic examination and corresponded to the areas of penetration by the biopsy instrument. Microscopic lesions that were indicative of testicular dysfunction were not found. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that testicular biopsy is a safe procedure in bulls. Testicular biopsy could possibly be used to further examine bulls that have less than satisfactory results for breeding soundness examinations.

Diagnosis and Treatment of Trichospirura leptostoma Infection in Common Marmosets (Callithrix jacchus).

Trichospirura leptostoma is a spirurid nematode that inhabits the pancreatic ducts of the common marmoset, Callithrix jacchus, and other New World primates. This parasite was diagnosed in members of this species at the Marmoset Research Center in Oak Ridge, Tennessee colony after their importation from multiple colonies outside the United States. Animals with few parasites in the pancreas had few tissue changes, whereas those with many parasites had moderate to severe fibrosis replacing exocrine tissue. Trichospirura leptostoma can cause chronic pancreatitis, resulting in subclinical to clinical pancreatic insufficiency and secondary malnutrition. Clinical signs of pancreatic insufficiency in affected marmosets included weight loss (despite good appetite) and increased fecal volume. Because earlier reports did not mention treatments, experimental therapeutic regimens were attempted to eliminate infections from the marmosets. In the clinical studies, different doses and treatment periods with ivermectin and fenbendazole were evaluated for therapeutic efficacy. Fenbendazole given daily at a dosage of 50 mg/kg of body weight by gavage for 14 days was the most effective treatment (PI 0.052). Control of the cockroach vector, as well as treatment of infected animals, is needed to prevent Trichospirura leptostoma-induced disease.

Use of ultrasound to monitor prenatal growth and development in the common marmoset (Callithrix jacchus).

The increasing use of non-human primates to study fetal development and neonatal management has necessitated the availability of fetuses of known gestational history. In this study, prenatal development and growth were investigated in the common marmoset (Callithrix jacchus) using ultrasound. The objectives of this study were: (1) to determine the accuracy of ultrasound for monitoring prenatal growth and development in common marmosets, (2) to determine if litter size influences prenatal growth trajectories, and (3) to assess growth discordancy among litter mates. Fifty pregnancies were monitored longitudinally using real-time abdominal sonography. During each examination the number of fetuses was recorded, and crown-rump length (CRL) and biparietal diameter (BPD) were measured. The results indicate that ultrasound is a reliable method for observation of gross morphological changes during prenatal development in this species. Measures of CRL and BPD taken early in gestation using ultrasound were in agreement with those from gross specimens. Triplets were significantly (P < 0.05) smaller than twins for both BPD and CRL. No significant relationship was found between litter size and within litter variation in CRL or BPD. This study is the first longitudinal investigation of prenatal growth and development in C. jacchus. The observations from this study will be of use for determining approximate gestational age of fetuses, as well as providing guidelines for routine monitoring of pregnancy in this species. © 1995 Wiley-Liss, Inc.

Infant-care behavior of mothers and fathers in a communal-care primate, the cotton-top tamarin (Saguinus oedipus).

Infant care behavior was examined for 35 litters of 23 different groups of captive cotton-top tamarins. The behavior of mothers and fathers was compared to determine the effects of infant number and helper presence on each parent and to determine whether parents differed in responsiveness to infants. For young infants (1-4 weeks), the contribution of fathers to infant carrying was negatively correlated with helpers' contribution; as helpers carried more, fathers carried less. In contrast, the mothers' contribution to infant carrying was unaffected by helper contribution. Mothers carried older infants (5-8 weeks) less than did fathers, regardless of infant number or helper contribution. Fathers and mothers were equally likely to retrieve a non-harassed infant; however, fathers were more likely than mothers to retrieve an infant being harassed. These results suggest that fathers are more responsive to infants than are mothers. Mothers may limit their involvement in infant care, as has been proposed by field results, due to an energetically demanding reproductive strategy.

Breeding performance of captive-born cotton-top tamarin (Saguinus oedipus) females: Proposed explanations for colony differences.

Successful breeding by captive-born Saguinus oedipus females has now been reported from several colonies, but a marked parity effect (ie, differences in infant survival with the number of litters produced) is usually observed; survival of infants of primiparous females is extremely low but increases with subsequent litters. This parity effect was not observed in the captive-born breeding females in the Oak Ridge Associated Universities colony, with survival of offspring of primiparous females at 58.6%. Two explanations are proposed for this difference: (1) pairing with a male having previous experience in siring and rearing offspring may improve infant survival for primiparous females; and (2) postponing mating to a later age may increase infant survival for primiparous mothers.

Comparison of infant care in family groups of the common marmoset (Callithrix jacchus) and the cotton-top tamarin (Saguinus oedipus).

The involvement of parents and siblings in infant care in similarly composed groups of common marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus) was compared during the infants' first 8 weeks of life. The results indicate an earlier infant independence in C. jacchus than in S. oedipus due primarily to a more frequent rejection of carried infants in C. jacchus. There was no species difference in extent of maternal involvment in carrying infants. However, S. oedipus fathers carried infants significantly more often during weeks 5-8 than did C. jacchus fathers. Siblings were generally more involved in infant care at an earlier infant age in C. jacchus than in S. oedipus.

Effects of sibling-rearing experience on future reproductive success in two species of callitrichidae.

The survival rate for offspring of mothers who either had or did not have previous experience rearing younger siblings was compared in two callitrichid species, Callithrix jacchus and Saguinus oedipus. Offspring of mothers with sibling-rearing experience had a higher survival percentage than offspring of inexperienced mothers in both species. While 50-60% of offspring of inexperienced C. jacchus mothers survived, no offspring of inexperienced S. oedipus mothers survived. The results suggest that sibling-rearing experience is necessary for adequate maternal behavior in S. oedipus, but not necessary to the development of maternal behavior in C. jacchus. Effects of previous sibling-rearing experience of S. oedipus fathers on offspring survival were also examined. Whether the father had rearing experience was not related to the survival of their offspring.