The evolutionary history of dogs in the Americas.

Dogs were present in the Americas before the arrival of European colonists, but the origin and fate of these precontact dogs are largely unknown. We sequenced 71 mitochondrial and 7 nuclear genomes from ancient North American and Siberian dogs from time frames spanning ~9000 years. Our analysis indicates that American dogs were not derived from North American wolves. Instead, American dogs form a monophyletic lineage that likely originated in Siberia and dispersed into the Americas alongside people. After the arrival of Europeans, native American dogs almost completely disappeared, leaving a minimal genetic legacy in modern dog populations. The closest detectable extant lineage to precontact American dogs is the canine transmissible venereal tumor, a contagious cancer clone derived from an individual dog that lived up to 8000 years ago.

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

GENITAL TRACT SCREENING FINDS WIDESPREAD INFECTION WITH MUSTELID GAMMAHERPESVIRUS 1 IN THE EUROPEAN BADGER ( MELES MELES).

: Sexually transmitted diseases (STDs) can be important drivers of population dynamics because of their negative effects on reproduction. However, screening for STDs, especially in wildlife populations, is widely neglected. Using the promiscuous, polygynandrous European badger ( Meles meles) as a model, we investigated the presence and prevalence of herpesviruses (HVs) in a wild, high-density population and assessed potential differences in somatic fitness and female reproductive condition between infected and uninfected individuals. We collected n=98 genital swabs from 71 females (51 adults and 20 cubs) and 27 males (26 adults and 1 cub) during spring and summer 2015. Using a PCR specific for a mustelid α-HV, all genital-swab samples tested negative. In a panherpes PCR, a γ-HV was found in 55% (54/98; 39 adults and 15 cubs), identified as mustelid gammaherpesvirus 1 (MusGHV-1) using DNA sequencing. This contrasts with the results of a previous study, which reported MusGHV-1 in 98% (354/361) of blood samples taken from 218 badgers in the same population using PCR. The detection of MusHV-1 in the female reproductive tract strongly indicates the potential for a horizontal and, likely also a vertical, route of transmission. Our results suggest a potential linkage of genital HVs and impaired future reproductive success in females, but because reproductive failure can have many reasons in badgers, the causative link of this negative relationship remains to be investigated.

Seropositivity to Sarcocystis infection of llamas correlates with breeding practices.

Production of llama (Lama glama) meat in rural communities of the Andean regions is largely affected by Sarcocystis spp. infection. Macroscopic cysts develop in muscles as a consequence of S. aucheniae parasitism, often resulting in meat downgrade or condemnation. Llama meat production is informal in Argentina but has broad perspectives for improvement, and would significantly benefit from the development of standardized control methodologies. This work analyzes whether the presence of anti-Sarcocystis spp. antibodies in llamas is influenced by factors such as geographic region and/or herd management practices. To this aim, an indirect ELISA was set up based on a ~23kDa soluble immunogenic protein fraction (Sa23), isolated from S. aucheniae macrocysts (Sa23-iELISA). Serum samples (n=507) were collected from llamas bred under three different conditions: (i) with no sanitation controls and in the presence of pastoral dogs by small producers of different localities of the Argentine Puna (Group I, n=237); (ii) with sanitation controls and no pastoral dogs, in fenced fields of an experimental agricultural station in the Argentine Puna (Group II, n=167); and (iii) with sanitation controls and no pastoral dogs in fenced fields of farms of the humid Pampas (Group III, n=103). Results of the Sa23-iELISA were expressed as percentages of positivity with respect to a reference Sarcocystis-positive serum. Notably, the percentage of sera that fell above the cut-off (31.5% positivity) in group (i) was significantly higher (p<0.001) than those of groups (ii) and (iii) (50% vs 23% and 26%, respectively). These results indicate that herd management practices constitute a critical risk factor for sarcocystiosis in llamas. Differences in these practices include feeding of dogs with raw Sarcocystis-infected llama meat, with the consequent maintenance of the parasite life cycle by the contamination of pastures and water with fecal-derived infective oocysts/sporocysts. Additionally, the itinerancy of llama herds in search for pastures and water sources possibly exposes animals to a higher number of infective foci. On the other hand, percentages of seropositive llamas kept under controlled conditions in the Puna or the humid Pampas were not significantly different, suggesting that climate, altitude, and/or pasture characteristics do not influence Sarcocystis-infection. Male gender and older age of llamas were found to be propensity factors for sarcocystiosis in llamas bred in La Puna under controlled conditions. Availability of diagnostic tools, as well as increased knowledge on the parasite and its epidemiology, will allow the design of control strategies for SAC sarcocystiosis.

Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: Evidence of Trichomonadidae infections.

Sexually transmitted diseases (STDs) can persist endemically, are known to cause sterility and infant mortality in humans, and could have similar impacts in wildlife populations. African apes (i.e., chimpanzees, bonobos, and to a lesser extent gorillas) show multi-male mating behavior that could offer opportunities for STD transmission, yet little is known about the prevalence and impact of STDs in this endangered primate group. We used serology and PCR-based detection methods to screen biological samples from wild and orphaned eastern chimpanzees and gorillas (N = 172 individuals, including adults, and juveniles) for four classes of pathogens that either commonly cause human STDs or were previously detected in captive apes: trichomonads, Chlamydia spp., Treponema pallidum (syphilis and yaws), and papillomaviruses. Based on results from prior modeling and comparative research, we expected STD prevalence to be highest in females versus males and in sexually mature versus immature individuals. All samples were negative for Chlamydia, Treponema pallidum, and papillomaviruses; however, a high percentage of wild chimpanzee urine and fecal samples showed evidence of trichomonads (protozoa). Analysis revealed that females were more likely than males to have positive urine-but not fecal-samples; however, there was no evidence of age (sexual maturity) differences in infection status. Sequence analysis of chimpanzee trichomonad samples revealed a close relationship to previously described trichomonads within the genus Tetratrichomonas. Phylogenetic comparisons to archived sequences from multiple vertebrate hosts suggests that many of the chimpanzee parasites from our study are likely transmitted via fecal-oral contact, but the transmission of some Tetratrichomonas sequence-types remains unknown and could include sexual contact. Our work emphasizes that only a fraction of infectious agents affecting wild apes are presently known to science, and that further work on great ape STDs could offer insights for the management of endangered great apes and for understanding human STD origins.

Experimental infection with equid herpesvirus 3 in seronegative and seropositive mares.

Equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), has been recognized as an economically significant venereal disease for years. However, no infection models on the natural host have been established. In order to set up an experimental infection protocol, seronegative and seropositive mares were topically inoculated in the perineal region with 4 × 10(6)TCID(50)/ml of EHV-3. Clinical signs were then evaluated by means of a designed scoring system, and body temperature was recorded daily. Virological, and serological studies were also performed. Typical ECE lesions, with clinical scores of 90, 92, 160 and 172, were observed in the four seronegative animals. Only mild ECE lesions were observed in the two seropositive mares, being the clinical scores 53 and 41. Both groups of mares shed the virus, but the duration of virus shedding was shorter and its intensity was lower in seropositive mares than in seronegative ones. Moreover, EHV-3 antibody response was detected in both seronegative and seropositive mares after experimental infection and re-infection, being more moderate in seropositive ones. As a conclusion, EHV-3 infection of mares was experimentally achieved in a reproducible manner. The typical lesions of ECE were observed after topical EHV-3 infection in seronegative mares, in association with virus excretion and neutralizing antibody kinetics.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis / Produção e caracterização de anticorpos monoespecíficos contra Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Para a produção de anticorpos monoclonais contra Campylobacter fetus subsp. venerealis foram utilizadas as linhagens de células de mieloma Sp2/0-Ag14 e células de baço de camundongos BALB/c imunizados com sonicado de C. fetus subsp. venerealis NCTC 10354. A detecção dos anticorpos monoclonais foi realizada por ELISA indireto utilizando antígeno sonicado de C. fetus subsp. venerealis NCTC 10354. A clonagem foi realizada por diluição limitante e os clones foram caracterizados por ELISA indireto utilizando um painel de bactérias escolhidas em função da prevalência e habitats: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp. fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352 e Arcobacter skirrowii LMG 6621; e no "western blotting" utilizando antígenos sonicados de C. fetus subsp. venerealis NCTC 10354 e C. sputorum biovar sputorum LMG 6647. Foram obtidos 15 clones produtores de anticorpos anti- C. fetus subsp. venerealis das classes IgM (1) e IgG (14). Quatro clones dentre os 15 clones obtidos foram produtores de anticorpos monoclonais espécie-específicos: dois clones reagiram com maior especificidade contra C. fetus subsp. venerealis NCTC 10354 e dois clones reagiram com maior especificidade contra C. fetus subsp. fetus ADRI 1812. Nenhum dos clones reagiu contra C. sputorum biovar sputorum LMG 6647, comprovando a especificidade dos anticorpos monoclonais testados. Todos os clones reconheceram uma proteína de massa molecular de aproximadamente 148 kDa no sonicado de C. fetus subsp. venerealis NCTC 10354.

Campilobacteriose genital bovina e tricomonose genital bovina: epidemiologia, diagnóstico e controle / Bovine genital campylobacteriosis and bovine genital trichomonosis: epidemiology, diagnosis and control

A presente atualização trata de duas das mais importantes doenças sexualmente transmitidas de bovinos, a campilobacteriose genital bovina e a tricomonose genital bovina. São abordados aspectos relacionados à epidemiologia destas doenças, principalmente em relação a sua distribuição no Brasil. Também são revisados aspectos importantes de diagnóstico, incluindo as técnicas e interpretação dos resultados, além de medidas de controle para ambas as doenças.

The present update deals with two of the most important sexually transmitted diseases of cattle: bovine genital campylobacteriosis and bovine genital trichomonosis. Epidemiological aspects, mainly their distribution in Brazil, alongside with their diagnosis in cattle are presented and commented. The main points in their diagnoses, including the description of the techniques and the interpretation of the results are also reviewed. Finally the control and prevention of both diseases are discussed.

Quantitative analysis of campylobacter fetus venerealis adhesion to bovine reproductive tract cell cultures / Análise quantitativa da adesão de campylobacter fetus venerealis em culturas de células do aparelho reprodutor bovino

Campylobacter fetus is the etiological agent of bovine genital campylobacteriosis, a sexually transmitted disease which is associated with reproductive losses in bovines. Campylobacter colonizes the vagina and the uterus and then infects the epithelial cells of the endometrium. The objective of this work was to develop an ex vivo model to quantify the adhesion of Campylobacter to its natural specific target cells; this is a key step for the establishment of infection and studies regarding the adherence and cytotoxicity on the natural host cells are not available. The assays were carried out by seeding Campylobacter fetus venerealis on bovine vaginal and uterine epithelial cell cultures. HeLa cells were used as control. Bacterial adhesion was corroborated by optical microscopy and determination of the percentage of adherent bacteria was performed on immunochemically-stained slides. Results are presented as percentage of cells with adherent Campylobacter and as number of bacteria per cell. In comparison to the control HeLa cells, the statistical analysis revealed that primary cultures show a higher percentage of infected cells and a lower variation of the evaluated parameters. This primary culture model might be useful for studies on cytopathogenicity and adhesion of different field strains of Campylobacter fetus.

Campylobacter fetus é o agente etiológico da campilobacteriose genital bovina, uma doença sexualmente transmissível que está associada com perdas reprodutivas em bovinos. Campylobacter coloniza a vagina e o útero e então infecta as células epiteliais do endométrio. O objetivo deste trabalho foi desenvolver um modelo ex vivo para quantificar a adesão de Campylobacter às células-alvo naturais específicas; este é um passo fundamental para o estabelecimento da infecção e estudos acerca da adesão e citotoxicidade sobre as células do hospedeiro natural não estão disponíveis. Os ensaios foram realizados a través da semeadura de Campylobacter fetus venerealis em culturas celulares epiteliais vaginais e uterinas.Células HeLa foram utilizadas como controle.A aderência bacteriana foi confirmada por microscopia óptica e a determinação da porcentagem de bactérias aderidas foi realizada em lâminas tingidas imunoquimicamente. Os resultados são apresentados como porcentagem de células com Campylobacter aderente e como o número de bactérias por células. Em comparação com as células HeLa controle, a análise estatística revelou que as culturas primárias mostram uma maior porcentagem de células infectadas e uma menor variação dos parâmetros avaliados. Este modelo de cultura primária pode ser útil para estudos sobre citopatogenicidade e adesão de diferentes cepas de campo de Campylobacter fetus.

Clinical utility of a life quality score in dogs with canine transmissible venereal tumor treated by vincristine chemotherapy / Utilidade clínica de escala de qualidade de vida durante o tratamento quimioterápico de cães com tumor venéreo transmissível canino (TVTC) com vincristina

The clinical utility of a life quality score during vincristine chemotherapy of dogs with canine transmissible venereal tumor (CTVT) was investigated using 93 tumor-bearing dogs in this prospective study. At diagnosis, clinical data and the performance status were evaluated according to a modified Karnofsky score (CKS) adapted for dogs. The animals were treated with vincristine sulphate (0.025mg/kg) at weekly intervals until the tumor had macroscopically disappeared. The time until the first adverse event and death were recorded. In a pilot study, CKS revealed a high inter-observer concordance (kappa=0.735; weighted kappa=0.835). CKS permitted a detailed monitoring of adverse effects during therapy. Cox regressions showed that low performance score and reduced body weight were independent predictive factors for death during chemotherapy. The modified Karnofsky performance score is a simple and reproducible clinical instrument, able to estimate patient outcome after treatment of CTVT.

Investigou-se a utilidade clínica de uma escala de qualidade de vida durante o tratamento quimioterápico de cães com tumor venéreo transmissível canino (TVTC) em 93 animais com TVTC que compuseram este estudo prospectivo. Ao diagnóstico, foram avaliados os dados clínicos e o escore de qualidade de vida adaptado para cães (CKS). Os animais foram tratados com sulfato de vincristina (0.025mg/kg) semanalmente até o desaparecimento macroscópico do tumor. Foram anotados o tempo até o aparecimento do primeiro evento adverso e a morte. Em estudo piloto do CKS, alta concordância entre os observadores foi demonstrada (kappa = 0.735; weighted kappa = 0.835). O CKS permitiu um detalhado monitoramento dos eventos adversos durante a terapia. A regressão Cox multivariada demonstrou que o baixo escore de qualidade de vida e o reduzido peso corporal foram fatores preditivos independentes para o óbito durante a quimioterapia. A escala modificada de Karnofsky é um instrumento clínico simples e reproduzível, hábil em estimar os efeitos do tratamento no TVTC.

Identification of canine transmissible venereal tumor cells using in situ polymerase chain reaction and the stable sequence of the long interspersed nuclear element.

Canine transmissible venereal tumor (CTVT) is a unique tumor that can be transplanted across the major histocompatibility complex (MHC) barrier by viable tumor cells. In dogs, CTVT grows progressively for a few months and then usually regresses spontaneously. A long interspersed nuclear element (LINE) insertion is found specifically and constantly in the 5' end of the CTVT cell c-myc gene, outside the first exon. The rearranged LINE-c-myc gene sequence has been used with polymerase chain reaction (PCR) to diagnose CTVT. However, in CTVT cells, the total length of the inserted LINE gene is not constant. In this experiment, variation in the inserted LINE gene was studied to determine which parts of the LINE sequence can be used as primers to identify CTVT cells with in situ PCR (IS PCR). The LINE gene was inserted between the TATA boxes in the promoter region of c-myc. In CTVT cells, deletions of different lengths are frequent in this gene. However, the 550-bp segment at the 5' end of the LINE-c-myc gene was stable. Thus, primers were designed to cover the stable 0.55-kb segment from the 5' end outside the first exon of the c-myc gene to the 5' end of LINE gene stable segment. With these primers and IS PCR, individual CTVT cells in formalin-fixed tissue sections and CTVT cultures were identified. Cells from other canine tumors were negative for this gene. In addition, the CTVT-specific, 0.55-kb segment was not found in any spindle-shaped cells from progressive or regressive phase CTVT. The IS PCR technique also did not detect any positive spindle-shaped cells in CTVT cell cultures. Thus, fibroblastic terminal differentiation is less likely to be a mechanism for spontaneous regression of CTVT cells.

Cancer selection.

Cancers are often thought to be selectively neutral. This is because most of the individuals that they kill are post-reproductive. Some cancers, however, kill the young and so select for anticancer adaptations that reduce the chance of death. These adaptations could reduce the somatic mutation rate or the selective value of a mutant clone of cells, or increase the number of stages required for neoplasia. New theory predicts that cancer selection--selection to prevent or postpone deaths due to cancer--should be especially important as animals evolve new morphologies or larger, longer-lived bodies, and might account for some of the differences in the causes of cancer between mice and men.

Evaluation of the field application of PCR in the eradication of contagious equine metritis from Japan.

The effectiveness of the polymerase chain reaction (PCR) as a field application test for the eradication of contagious equine metritis (CEM) was evaluated. Seven-thousands five-hundred and thirty-four genital swabs were collected from 4,026 Thoroughbred broodmares and stallions in Japan to test "high risk" horses as well as for general surveillance testing from 1998 to 2001. Bacterial isolation as well as PCR testing of original specimens and cultured specimens was performed for detection of Taylorella equigenitalis from genital swabs. As a result, T. equigenitalis was detected in 12 mares and 1 stallion by PCR, although the bacteria were isolated from only 2 of the PCR-positive mares. CEM-infected and carrier horses were treated by a combination of chemotherapy and surgery. Subsequent follow-up testing over a 3-year period did not detect T. equigenitalis. It was demonstrated that PCR testing was more sensitive than isolation as a method for the detection of T. equigenitalis from genital swabs of horses in the field. It was therefore suggested that a combination of PCR testing and treatment were useful measures in the eradication of CEM from Japan.

Heat shock proteins in canine transmissible venereal tumor.

SDS-PAGE, Western blot analysis and immunohistochemical staining were used to detect heat shock proteins (HSPs) 60, 70 and 90 in canine transmissible venereal tumor (CTVT). Tissues tested for HSPs included: (1) tissues from different growth phases of CTVT tumors artificially induced in dogs; (2) tissues from other canine tumors; (3) normal dog tissues. Our results indicate that HSP 60 was consistently higher in CTVT cells in regressing phase than those in progressing phase. However, no detectable antibody response specific to the tested HSPs was found in the sera from CTVT-laden dogs in different growth phases. Although levels of the HSPs were all detectable in CTVT cells, only 60 and 70 were higher in CTVT cells than in normal tissues. In addition, none of the HSPs were detected in cells from five other canine tumors. These data suggest that canine HSP 60 and 70 are potential markers for CTVT and HSP 60 is appear to be involved in CTVT regression.PCR was used to confirm the existence of CTVT cells using primers designed to cover the sequence between the 5' end of c-myc near the first exon and the 3' end outside the LINE gene. Only CTVT samples were positive for this sequence; samples from other tumors and normal tissues were negative. The sequenced PCR products indicated that CTVT from Taiwan and other countries exhibited over 98% sequence homology. This reconfirms that, worldwide, all CTVT cells are very similar.

Proliferation characteristics of canine transmissible venereal tumor.

Canine transmissible venereal tumor (CTVT) grows progressively (P-phase) in the host and then spontaneously regresses (R-phase). The mechanisms behind the transition from the P-to R-phases are not well understood. In this study, in order to determine the proliferation characteristics of CTVT, we evaluated telomerase activity and enumerated nuclear organizing regions (AgNOR) and proliferating cell nuclear antigen (PCNA). It was found that CTVT cells from the P-and R-phases were both positive for telomerase activity, although it was lower in the R-phase. Evaluations of telomerase activity should take into account the stage of mitosis. Although, in the majority of cases, telomerase activity can be used to differentiate between benign and malignant tumors in dogs, other factors or markers should also be used to obtain accurate diagnoses. The PCNA-positive rate and the number and area of AgNOR per cell increased much more in the P-phase than the R-phase. However, the AgNOR values were always higher. Thus, the AgNOR count can be used to distinguish the P-and R-phases of CTVT. In addition, mitotic figures were much higher in number in the P-phase as compared to the R-phase. We believe that, during spontaneous regression of CTVT cells, slow tumor cell proliferation must contribute to the decrease in tumor size. However, shortening of tumor cell telomeres is not directly involved in this process. Other factors, such as expression of MHC antigens on CTVT cells, humoral immunity, cytokines released by the inflammatory cells and, especially, tumor infiltrating lymphocytes may contribute to CTVT regression.

Use of exfoliative cytology for diagnosis of transmissible venereal tumour and controlling the recovery period in the bitch.

Transmissible venereal tumour (TVT) is a rare vaginal tumour that can be treated surgically or cryosurgically as well as by radiotherapy or chemotherapy. Vincristine has been found to be very effective for treating TVT. Since vaginal secretion or discharge may contain neoplastic cells, the cytological identification of TVT cells is possible. The present study was carried out in 12 bitches. Vaginal smears were obtained with cotton swab from the anterior vagina and TVT suspected structures. The smears were stained according to Papanicolaou and assessed by light microscopy. Additionally the general condition of the patients was evaluated by haematological and radiographic examinations. In bitches with TVT vincristine sulphate was administered intravenously at weekly intervals. The total treatment period was three to six weeks until no atypical cells were found in the smear. This was the case after an average of 3.2 +/- 1.3 applications. Tumour masses became smaller and by this non-visible from the rima vulva after 4.2 +/- 0.7 applications. During the treatment, two of the 12 bitches (16.7%) suffered from vomiting and diarrhoea while three (25%) showed neutropenia. Twelve months after completion of treatment, the bitches were examined again and vaginal smears were taken in order to control the recovery process or a possible recurrence of TVT. No atypical cells were found in any vaginal smear. By this exfoliative cytology has proved to be a safe and easy method for TVT diagnosis and for observing the recovery process.

Changes in lymphokine-activated killer activity in peripheral blood lymphocytes from canine transmissible venereal sarcoma models.

Time course changes in anti-tumor activity induced in peripheral blood lymphocytes (PBL) with recombinant human interleukin-2 (rhIL-2) and phytohemagglutinin-P (PHA) were studied in dogs implanted with canine transmissible venereal sarcoma (CTVS) as a tumor-bearing model. The rhIL-2-dependent and PHA-dependent cultures allowed selective proliferation of lymphocytes expressing Thy-1 antigens. The lymphocytes acquired a prolonged anti-tumor activity against the CTVS cells, starting from 2 weeks after the culture, indicating generation of lymphokine-activated killer (LAK) cells. The LAK cells showed serial growth in rhIL-2-containing culture medium for at least a further 2 weeks without loss of the anti-tumor activity.

Clinico-pathological studies on the effect of different anti-neoplastic chemotherapy regimens on transmissible venereal tumours in dogs.

Thirty-two dogs affected with transmissible venereal tumour (TVT) were divided into three treatment groups. In group I vincristine sulphate at 0.025 mg/kg body weight, in group II vinblastine sulphate at 0.150 mg/kg body weight, and in group III vinblastine sulphate at 0.100 mg/kg body weight plus methotrexate at 0.35 mg/kg body weight were given intravenously at weekly intervals. Biopsies were performed on days 0, 3, 7 and 14. The tissues were preserved in 10% neutral buffered formalin and processed routinely for haematoxylin and eosin staining. Histopathologically, the untreated TVT was characterized by sheets or bundles of mostly rounded cells having a large, highly basophilic nucleus with a prominent, highly basophilic necleolus. Both vincristine and vinblastine primarily affected the nuclei of neoplastic cells, causing condensation, karyorrhexis and karyolysis within 3 days of chemotherapy. The regressing tumour mass showed marked infiltration by lymphocytes, lymphoblasts and macrophages by day 7. There was nearly complete regression of the tumour by day 14, as shown by the almost complete loss of neoplastic cells, with fibrous tissue substitution. However, in group III, the changes occurred more slowly and more injections were needed for complete regression. In both groups I and II, 11/12 of the animals responded completely to the chemotherapy within 3 weeks, while in group III, 6/8 of the dogs responded to the treatment by 21-28 days.