Triatomine vectors of Trypanosoma cruzi in an endemic area for Chagas disease in Northeast Brazil.

BACKGROUND: We assessed the distribution of triatomines in an endemic area for Chagas disease. METHODS: This retrospective study used secondary data extracted from the Official System of the National Chagas Disease Control Program (Sistema Oficial do Programa Nacional de Controle da Doença de Chagas - SisPCDCh). RESULTS: A total of 7,257 (725.7 ± 221.7 per year) specimens were collected from 2013 to 2022. Most of them (6,792; 93.6%) were collected in the intradomicile and 465 (6.4%) in the peridomicile. A total of 513 (7.1%) triatomines tested positive for the presence of trypomastigote forms, similar to Trypanosoma cruzi. CONCLUSIONS: The spatial analysis revealed a heterogeneous distribution of triatomines across different municipalities.

Triatomine vectors of Trypanosoma cruzi in an endemic area for Chagas disease in Northeast Brazil

ABSTRACT Background: We assessed the distribution of triatomines in an endemic area for Chagas disease. Methods: This retrospective study used secondary data extracted from the Official System of the National Chagas Disease Control Program (Sistema Oficial do Programa Nacional de Controle da Doença de Chagas - SisPCDCh). Results: A total of 7,257 (725.7 ± 221.7 per year) specimens were collected from 2013 to 2022. Most of them (6,792; 93.6%) were collected in the intradomicile and 465 (6.4%) in the peridomicile. A total of 513 (7.1%) triatomines tested positive for the presence of trypomastigote forms, similar to Trypanosoma cruzi. Conclusions: The spatial analysis revealed a heterogeneous distribution of triatomines across different municipalities.

Canine visceral leishmaniasis: risk factors and spatial analysis in an endemic area of Northeastern Brazil.

Canine visceral leishmaniasis is a disease caused by the protozoon Leishmania infantum, an important agent of zoonotic concern. In this study we determined the seroprevalence, risk factors and spatial distribution of infection by L. infantum among dogs in the Pajeú microregion of the Sertão region of Pernambuco, Brazil. Canine serum samples (n = 247) were tested using the Dual Path Platform (DPP®) rapid screening test and ELISA/S7® confirmatory test; and risk factors were assessed through univariate analysis and logistical regression. The spatial distribution of reactive dogs was analyzed by drawing a map using QGIS. Seroprevalence of 13.7% (34/247) was detected, with cases predominating in the municipality of Tabira (26.4%; 9/34). Age above 10 years was considered to be a risk factor associated with the presence of anti-L. infantum antibodies. The high overall prevalence and spatial distribution of positive cases showed wide dispersion of reagents dogs in the study area. Therefore, preventive measures are needed in order to reduce the risk of infection for animals and humans.

Molecular detection of vector-borne pathogens in cats tested for FIV and FeLV.

The aim of this study was to detect molecularly vector borne pathogens (VBPs) in domiciled cats tested for Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV). Blood samples (n = 119) were analyzed microscopically and molecularly through PCR and sequenced for the detection of the following pathogens: piroplasmids., Bartonella henselae, Cytauxzoon felis, Ehrlichia canis, Leishmania spp., hemotropic Mycoplasma spp., Trypanosoma spp., and Ricketssia spp. Animals were also serological assessed for detection of antibodies against FIV and FeLV. Out of all animals, 20.16% (24/119) tested positive for at least one VBPs at molecular examination. Conversely, no animal resulted positive at microscopic analysis. The most prevalent pathogen was hemotropic Mycoplasma haemofelis (8.40%; 10/119), followed by Candidatus Mycoplasma haemominutum (5.88%; 7/119), E. canis (5.04%; 6/119), C. felis (0.84%; 1/119) and B. henselae (0.84%; 1/119). One animal (0.84%; 1/119) was co-infected with. E. canis and B. henselae. A total of 5.88% (7/119) and 1.68% (2/119) tested positive for FIV and FeLV, respectively. Data of this study demonstrate that owned cats can be at risk of hemotropic Mycoplasma spp., E. canis, C. felis and B. henselae. Therefore, preventive measures against vectors of these pathogens should be implemented in order to reduce the risk of exposition and consequently infection. Additionally, aggressive behaviors among cats should be avoided, especially because hemotropic Mycoplasma spp. may be transmitted through the bite of animals.

Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil.

The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 µm (± 28.65 µm) and mean width of 19 µm (± 1.03 µm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Diversity of Eimeria (Apicomplexa: Eimeriidae) species and risk factors associated in natural infecting calves at the Southern Agreste Microregion in the State of Pernambuco, Brazil.

Eimeria species have importance to calves because of the economic losses. The aim of this study was to identify the species of Eimeria that affect calves and the risk factors associated with its natural infection. Fecal samples (387) were collected from dairy farms in the southern Agreste of Pernambuco. The feces were evaluated using the Gordon & Whitlock technique and were cultured in 2.5% potassium dichromate for sporulation of oocysts. Odds ratio (OR) were calculated to assess risk factors. Eimeria spp. were detected in 50.65% (196/387) of the samples. Eleven species were identified, being Eimeria bovis (26.64%; 548/2057), Eimeria zuernii (19.69%; 405/2057) and Eimeria ellipsoidalis (14.49%; 298/2057) those more frequent. Small herds (OR = 1.93), calves aged up to six months (OR = 2.12), absence of manure pit (OR = 7.52), fortnightly cleaning (OR = 4.71), collective calf pens (OR = 3.26), manual milking (OR = 2.16) and absence of veterinary care (OR = 2.28) were considered to be risk factors. The data revealed pathogenic species in more than 50% of the farms. Thus, the importance of adopting sanitary measures to reduce the spread of these protozoa in herds should be done, because of economic losses associated with its infection.

Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil / Estudo larvoscópico de Dictyocaulus sp. em fezes de bovinos de corte do nordeste do Brasil

Abstract The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 μm (± 28.65 μm) and mean width of 19 μm (± 1.03 μm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Resumo O parasito pulmonar Dictyocaulus viviparus tem um papel importante na saúde e produtividade bovina em todo o mundo, uma vez que infecções podem levar a perdas econômicas. Além de sua importância, poucos estudos que investigam aspectos epidemiológicos das infecções por esse parasito têm sido realizados. Objetivou-se, nesta pesquisa, relatar a ocorrência de infecção por nematódeos pulmonares em rebanhos bovinos de corte, criados em uma importante área de produção pecuária, na região Nordeste do Brasil. De setembro de 2020 a agosto de 2021, foram coletadas, mensalmente, amostras fecais (n = 493) de 46 bovinos de corte. De todos os animais avaliados, larvas de nematódeos pulmonares foram detectadas em 8,7% (4/46). Os animais não apresentaram nenhum sinal clínico sugestivo de infecção por parasitos pulmonares. Vinte larvas foram recuperadas, com o número mínimo (n = 1) detectado em outubro e dezembro, e o número máximo (n = 13) em novembro. Apresentavam comprimento médio de 363 μm (± 28,65 μm), largura média de 19 μm (±1,03 μm) e eram morfologicamente semelhantes a Dictyocaulus sp.. Este estudo relata a ocorrência desse parasito nesta área. Logo, os veterinários locais devem estar atentos à inclusão desse parasito no diagnóstico diferencial de outras infecções respiratórias em bovinos de corte.

Larvicidal activity of Caesalpinia ferrea Mart. and Lippia origanoides Cham. Against Aedes aegypti (LINNAEUS, 1762) (Diptera: Culicidae)

The control of Aedes aegypti has been considered one of the most important public health challenges worldwide. Chemical compounds have long been used for this purpose, but resistance to these molecules has also increased. Therefore, over the last few years several studies have focused on the development of alternative tools, particularly those based on plant metabolites. The purpose of this study was to assess the larvicidal activity of Caesalpinia ferrea and Lippia origanoides against Ae. aegypti. Larvae (L3) of Ae. aegypti Liverpool and Rockefeller strains, as well as of the Recife population were exposed to different concentrations of C. ferrea (ranging from 13.1 to 105 mg/mL) and L. origanoides (ranging from 16.3 to 130 mg/mL), and the mortality rate was evaluated up to 48 hours after the beginning of the experiment. All tested groups and ontrol group were quadruplicated. For C. ferrea, mortality ranged from 42.5% to 100% for Ae. aegypti Liverpool strain, from 67% to 100% for Ae. aegypti Rockfeller strain, and 57% to 100% for Ae. aegypti Recife population after 48 hours of larval exposure. For L. origanoides, the larvicidal activity ranged from 75% to 100% for Ae. aegypti Liverpool strain, from 61.5% to 100% for Ae. aegypti Rockfeller strain, and from 60.5% to 100% for Ae. aegypti Recife population. The hydro ethanol extract of C. ferrea and L. origanoides presented larvicidal activity against Ae. aegypti.

Sandflies in an endemic area for Visceral Leishmaniasis in Northeastern Brazil / Flebotomíneos em uma área endêmica para Leishmaniose Visceral no Nordeste do Brasil

Abstract Phlebotomines of the genus Lutzomyia are important vector for species of Leishmania, the etiological agent of leishmaniasis. The aim of this study was to assess the diversity of sandflies in an endemic area for Visceral Leishmaniasis (VL). Sampling was performed in four localities on the coast of the municipality of Goiana, state of Pernambuco, Northeastern Brazil. Specimens were collected monthly on three consecutive nights, from November 2017 to October 2018. CDC light traps were installed at a height of 1.5 m above the ground, in two ecotypes (i.e. tree and animal facility), both in peridomestic areas. A total of 5,205 sandflies were collected, among which the species Lutzomyia longipalpis (99.85%; 5,197/5,205) was the most common, followed by Lutzomyia evandroi (0.13%; 07/5,205) and Lutzomyia whitmani (0.02%; 01/5,205). Specimens of L. longipalpis were collected throughout the study period and most of them were found near to the animal facilities. The findings from this research indicate that vectors for Leishmania infantum are present in the study area throughout the year, along with occurrences of vectors relating to Cutaneous Leishmaniasis (CL). Preventive vector control measures need to be implemented in Goiana to reduce the risk of infection for the human and animal populations.

Resumo Flebotomíneos do gênero Lutzomyia são importantes vetores de espécies de Leishmania, agente etiológico das leishmanioses. O objetivo deste estudo foi avaliar a diversidade de flebotomíneos em área endêmica para Leishmaniose Visceral (LV). As coletas foram realizadas em quatro localidades do litoral do município de Goiana, Pernambuco, Nordeste do Brasil. Os espécimes foram coletados mensalmente em três noites consecutivas de novembro de 2017 a outubro de 2018. Foram instaladas armadilhas luminosas modelo CDC a uma altura de 1,5 m acima do solo em dois ecótopos (ex. árvore e instalações de animais), ambos em áreas peridomiciliares. Um total de 5.205 flebotomíneos foi coletado, sendo Lutzomyia longipalpis (99,85%; 5.197/5.205) a mais comum, seguida por Lutzomyia evandroi (0,13%; 07/5.205) e Lutzomyia whitmani (0,02%; 01/5.205). L. longipalpis foi coletada durante todo o período do estudo. A maioria dos espécimes foi detectada perto das instalações dos animais. Os achados deste estudo indicam a presença de vetores de Leishmania infantum na área avaliada ao longo do ano, bem como a ocorrência de vetores relacionados à Leishmaniose Cutânea (LC). Medidas preventivas de controle vetorial devem ser implementadas em Goiana para reduzir o risco de infecção à população humana e animal.

Spatial distribution of triatomine bugs in a Chagas disease endemic region in Brazil.

INTRODUCTION: Trypanosoma cruzi is the etiological agent of Chagas disease (CD), a zoonotic infection transmitted by triatomine bug vectors to human beings. Although the story of this parasitic infection was born in Brazil and here this has made major step forward information, the same cannot be said about the actual distribution of the triatomine vector in several areas of this country. The aim of this study was to assess the occurrence of triatomine species in an endemic region for CD in Northeastern Brazil. METHODS: A retrospective study was performed using data obtained from 2008 to 2017. All information was provided by the V Gerência Regional de Saúde of the state of Pernambuco. The spatial distribution of triatomine species was analyzed by drawing a map using the Quantum geographic information system. RESULTS: A total of 4,694 triatomine specimens (469.4 ± 221.2 per year) were collected during the period 2008-2017, with 94.5% (4,434/4,694) at the intradomicile and 5.5% (260/4,694) at peridomicile environment. Of all arthropods collected, 92.5% (4,340/4,694) and 7.5% (354/4,694) were adults and nymphs, respectively. The species most frequently detected were Panstrongylus lutzi (30.36%), Triatoma brasiliensis (26.12%), Triatoma pseudomaculata (22.43%), and Panstrongylus megistus (20.54%). CONCLUSIONS: These data contribute to a better understanding of the distribution of T. cruzi infection in the Northeastern region of Brazil. Preventive measures based on vector control should be implemented in the study area in order to reduce the burden this neglected tropical disease.

Sandflies in an endemic area for Visceral Leishmaniasis in Northeastern Brazil.

Phlebotomines of the genus Lutzomyia are important vector for species of Leishmania, the etiological agent of leishmaniasis. The aim of this study was to assess the diversity of sandflies in an endemic area for Visceral Leishmaniasis (VL). Sampling was performed in four localities on the coast of the municipality of Goiana, state of Pernambuco, Northeastern Brazil. Specimens were collected monthly on three consecutive nights, from November 2017 to October 2018. CDC light traps were installed at a height of 1.5 m above the ground, in two ecotypes (i.e. tree and animal facility), both in peridomestic areas. A total of 5,205 sandflies were collected, among which the species Lutzomyia longipalpis (99.85%; 5,197/5,205) was the most common, followed by Lutzomyia evandroi (0.13%; 07/5,205) and Lutzomyia whitmani (0.02%; 01/5,205). Specimens of L. longipalpis were collected throughout the study period and most of them were found near to the animal facilities. The findings from this research indicate that vectors for Leishmania infantum are present in the study area throughout the year, along with occurrences of vectors relating to Cutaneous Leishmaniasis (CL). Preventive vector control measures need to be implemented in Goiana to reduce the risk of infection for the human and animal populations.

Spatial distribution of Aedes aegypti (Diptera: Culicidae) in vulnerable areas for the transmission of arboviruses.

INTRODUCTION: Areas at risk of transmission of arboviruses have been monitored using ovitraps. This study aimed to evaluate the spatial distribution of Aedes aegypti in vulnerable areas for the transmission of arboviruses and assess the influence of climatic conditions on the infestation of these culicids. METHODS: Ovitraps were installed in Agrestina, Pernambuco, Northeastern Brazil. RESULTS: Overall, 44,936 eggs were collected, and the indexes of infestation varied. Relative humidity was significantly associated with the infestations. CONCLUSIONS: Using ovitraps, entomologic indexes and analysis of climatic factors might be good strategies for monitoring vulnerable areas for the transmission of arboviruses.

Seroprevalence of Neospora caninum and Toxoplasma gondii in dogs from an urban area of North-eastern Brazil: a spatial approach.

INTRODUCTION: The present study aimed to assess the seroprevalence and spatial distribution of Neospora caninum and Toxoplasma gondii in dogs. METHODS: Blood samples (n = 241) were collected and analyzed for the presence of anti-N. caninum and anti-T. gondii antibodies. The spatial distribution was evaluated using kernel density estimation (KDE). RESULTS: Anti-N. caninum and anti-T. gondii antibodies were detected in 24.06% (58/241) and 9.54% (23/241) of samples, respectively. A heterogeneous spatial distribution of positive dogs was observed across the city. CONCLUSIONS: These data are pivotal for better understanding the dynamics of infection caused by these protozoa in the canine population.

Spatial distribution of triatomine bugs in a Chagas disease endemic region in Brazil

Abstract INTRODUCTION: Trypanosoma cruzi is the etiological agent of Chagas disease (CD), a zoonotic infection transmitted by triatomine bug vectors to human beings. Although the story of this parasitic infection was born in Brazil and here this has made major step forward information, the same cannot be said about the actual distribution of the triatomine vector in several areas of this country. The aim of this study was to assess the occurrence of triatomine species in an endemic region for CD in Northeastern Brazil. METHODS: A retrospective study was performed using data obtained from 2008 to 2017. All information was provided by the V Gerência Regional de Saúde of the state of Pernambuco. The spatial distribution of triatomine species was analyzed by drawing a map using the Quantum geographic information system. RESULTS: A total of 4,694 triatomine specimens (469.4 ± 221.2 per year) were collected during the period 2008-2017, with 94.5% (4,434/4,694) at the intradomicile and 5.5% (260/4,694) at peridomicile environment. Of all arthropods collected, 92.5% (4,340/4,694) and 7.5% (354/4,694) were adults and nymphs, respectively. The species most frequently detected were Panstrongylus lutzi (30.36%), Triatoma brasiliensis (26.12%), Triatoma pseudomaculata (22.43%), and Panstrongylus megistus (20.54%). CONCLUSIONS: These data contribute to a better understanding of the distribution of T. cruzi infection in the Northeastern region of Brazil. Preventive measures based on vector control should be implemented in the study area in order to reduce the burden this neglected tropical disease.

Spatial distribution of Aedes aegypti (Diptera: Culicidae) in vulnerable areas for the transmission of arboviruses

Abstract INTRODUCTION: Areas at risk of transmission of arboviruses have been monitored using ovitraps. This study aimed to evaluate the spatial distribution of Aedes aegypti in vulnerable areas for the transmission of arboviruses and assess the influence of climatic conditions on the infestation of these culicids. METHODS: Ovitraps were installed in Agrestina, Pernambuco, Northeastern Brazil. RESULTS: Overall, 44,936 eggs were collected, and the indexes of infestation varied. Relative humidity was significantly associated with the infestations. CONCLUSIONS: Using ovitraps, entomologic indexes and analysis of climatic factors might be good strategies for monitoring vulnerable areas for the transmission of arboviruses.

Seroprevalence of neospora caninum and toxoplasma gondii in dogs from an urban area of North-eastern Brazil: a spatial approach

Abstract INTRODUCTION: The present study aimed to assess the seroprevalence and spatial distribution of Neospora caninum and Toxoplasma gondii in dogs. METHODS: Blood samples (n = 241) were collected and analyzed for the presence of anti-N. caninum and anti-T. gondii antibodies. The spatial distribution was evaluated using kernel density estimation (KDE). RESULTS Anti-N. caninum and anti-T. gondii antibodies were detected in 24.06% (58/241) and 9.54% (23/241) of samples, respectively. A heterogeneous spatial distribution of positive dogs was observed across the city. CONCLUSIONS These data are pivotal for better understanding the dynamics of infection caused by these protozoa in the canine population.

Seroprevalence and spatial distribution of canine leishmaniasis in an endemic region in Brazil: how has the situation changed after 10 years?

INTRODUCTION: Herein, we assessed the seroprevalence and spatial distribution of Leishmania infantum in dogs in Garanhuns, Northeastern Brazil. METHODS: Sera samples (n = 242) were analyzed using an enzyme-linked immunosorbent assay (ELISA). The spatial distribution of dogs seropositive for anti-Leishmania infantum antibodies was evaluated using kernel density estimation. RESULTS: A total of 2.4% (6/242) of the animals were seropositive for anti-Leishmania infantum antibodies. The kernel map showed their distribution to be heterogeneous over the city, with a hotspot in the northeastern region. CONCLUSIONS: The reported data illustrate the circulation of parasites of the genus Leishmania in a canine population.

Seroprevalence and spatial distribution of canine leishmaniasis in an endemic region in Brazil: how has the situation changed after 10 years?

Abstract INTRODUCTION: Herein, we assessed the seroprevalence and spatial distribution of Leishmania infantum in dogs in Garanhuns, Northeastern Brazil. METHODS: Sera samples (n = 242) were analyzed using an enzyme-linked immunosorbent assay (ELISA). The spatial distribution of dogs seropositive for anti-Leishmania infantum antibodies was evaluated using kernel density estimation. RESULTS: A total of 2.4% (6/242) of the animals were seropositive for anti-Leishmania infantum antibodies. The kernel map showed their distribution to be heterogeneous over the city, with a hotspot in the northeastern region. CONCLUSIONS: The reported data illustrate the circulation of parasites of the genus Leishmania in a canine population.

Screening of Cercopithifilaria bainae and Hepatozoon canis in ticks collected from dogs of Northeastern Brazil.

Rhipicephalus sanguineus sensu lato (s.l.) is one of the most widespread ixodid ticks and is a competent vector of several vector-borne pathogens of veterinary and medical concern. For instance, this tick species transmits nematodes of the genus Cercopithifilaria and protozoa of the genus Hepatozoon to carnivores, including dogs. Here we investigated the occurrence of Cercopithifilaria spp. and Hepatozoon spp. in a population of ticks collected from naturally infested dogs living in rural areas of Northeastern Brazil. From August 2016 to June 2017, 758 tick specimens (mean ticks per month = 68.9 ± 71.4) were sampled from 75 dogs (mean ticks per dog = 10.11 ± 5.2) and dissected under a stereomicroscope in order to visualize Cercopithifilaria spp. larvae and Hepatozoon spp. oocysts and sporocysts. R. sanguineus s.l. was the only species collected, peaking in September (n = 273) and decreasing in February 2017 (n = 39). Different larval stages of Cercopithifilaria bainae were identified in 7 out of 758 (0.93%) ticks. In addition, 4 specimens (0.53%) were positive for oocysts and free sporocysts of Hepatozoon canis. The identity of both species of parasites was molecularly confirmed. These results account for the predominance of R. sanguineus (s.l.) in domestic dogs from rural locations of the study area, as well as for the presence C. bainae and H. canis in these tick populations.

Reliability of different methods of diagnosing intestinal parasites in horses

Parasitism by intestinal nematodes may cause serious injuries to equines affecting their performance, given that the parasites compete for food and cause irritation, internal bleeding and anaemia. The diagnostic methods currently available are not efficient in detecting all the species of parasites simultaneously, hampering reliable diagnosis. Therefore, the purpose of this study was to evaluate four diagnostic methods for detecting equine intestinal parasites in the southern hinterland of Pernambuco, Brazil. Fecal samples (n = 87) were collected and examined through flotation based methods (Willis-Mollay, modified centrifugal flotation and EPG/OPG) and sedimentation (Hoffman). Of the total number of samples examined, 77.0% (67/87) were positive by modified centrifugal flotation; 44.8% (39/87) were positive by the Willis-Mollay method; 34.5% (30/87) by the Hoffman method and 28.7% (25/87) by the EPG/ OPG method. The Kappa index indicated moderate concordance between the Willis-Mollay and modified centrifugal flotation techniques (K= 0.477); Willis-Mollay and EPG/OPG (K= 0.466); EPG/OPG and Hoffman (K= 0.425). In conclusion, modified centrifugal flotation presented high sensitivity for detection of parasites of the Strongylida order and Parascaris spp. It may, therefore, be used in association with the Willis-Mollay technique as a safe and accurate method of diagnosis.