Interventions Targeting Deep Tissue Lymph Nodes May Not Effectively Reduce the Risk of Salmonellosis from Ground Pork Consumption: A Quantitative Microbial Risk Assessment.

The inclusion of deep tissue lymph nodes (DTLNs) or nonvisceral lymph nodes contaminated with Salmonella in wholesale fresh ground pork (WFGP) production may pose risks to public health. To assess the relative contribution of DTLNs to human salmonellosis occurrence associated with ground pork consumption and to investigate potential critical control points in the slaughter-to-table continuum for the control of human salmonellosis in the United States, a quantitative microbial risk assessment (QMRA) model was established. The model predicted an average of 45 cases of salmonellosis (95% CI = [19, 71]) per 100,000 Americans annually due to WFGP consumption. Sensitivity analysis of all stochastic input variables showed that cooking temperature was the most influential parameter for reducing salmonellosis cases associated with WFGP meals, followed by storage temperature and Salmonella concentration on contaminated carcass surface before fabrication. The input variables were grouped to represent three main factors along the slaughter-to-table chain influencing Salmonella doses ingested via WFGP meals: DTLN-related factors, factors at processing other than DTLNs, and consumer-related factors. The evaluation of the impact of each group of factors by second-order Monte Carlo simulation showed that DTLN-related factors had the lowest impact on the risk estimate among the three groups of factors. These findings indicate that interventions to reduce Salmonella contamination in DTLNs or to remove DTLNs from WFGP products may be less critical for reducing human infections attributable to ground pork than improving consumers' cooking habits or interventions of carcass decontamination at processing.

An outcomes model to evaluate risks and benefits of Escherichia coli vaccination in beef cattle.

We developed a stochastic simulation model to evaluate the impact of Escherichia coli O157:H7 (O157) vaccination on key epidemiological outcomes. The model evaluated a reduction in the O157 prevalence in feedlot cattle as well as concentration in cattle feces due to vaccination. The impact of this reduction on outcomes at slaughter/harvest and consumption was evaluated by simulating the relationships between the O157 prevalence and concentration at various points in the ground beef supply chain. The uncertainty and variability associated with the O157 contamination was explicitly modeled in production, slaughter, and consumption modules. Our results show that vaccination can have a significant benefit with respect to relevant outcomes such as (1) the number of human O157 illnesses due to the consumption of ground beef, (2) the number of production lots with high O157 contamination levels, (3) the likelihood of detection by U.S. Department of Agriculture Food Safety and Inspection Service testing, and (4) the probability of multiple illnesses due to ground beef servings from the same lot. These results show that these outcomes are strongly impacted by preharvest vaccination. For example, if the vaccine is used so as to reduce the prevalence of E. coli shedding cattle by 80% and if all U.S. steers and heifers were vaccinated, the expected number of human illnesses from ground beef-associated O157 would be reduced almost 60%. If the vaccine is 60% or 40% effective, the illness rate would be reduced approximately 45% or 40%, respectively. The number of production lots (10,000-lb lots) with high O157 contamination levels (> 1000 servings) would be reduced by 96% if all steers and heifers received an 80% effective vaccine regimen. The analysis shows that resulting reduction in the number of shedding animals and the reduced concentration of E. coli on carcasses can combine to reduce human illnesses and cost to beef packers.

Quantitative risk from fluoroquinolone-resistant Salmonella and Campylobacter due to treatment of dairy heifers with enrofloxacin for bovine respiratory disease.

The objective of this study was to evaluate the human health impact of using fluoroquinolones to treat bovine respiratory disease (BRD) in dairy heifers less than 20 months of age. Specifically, this study quantified the probability of persistent symptoms in humans treated with a fluoroquinolone, for a fluoroquinolone-resistant Campylobacter, Salmonella, or multidrug-resistant (MDR) Salmonella infection acquired following the consumption of ground beef. To comply with a Food and Drug Administration requirement for approval of enrofloxacin use in dairy heifers, a binomial event tree was constructed following Food and Drug Administration guidance 152. Release was estimated from the slaughter of dairy cattle carrying fluoroquinolone-resistant bacteria attributed to the proposed use in dairy heifers. For exposure, human foodborne exposure to Campylobacter, Salmonella, and MDR Salmonella after consumption of ground beef was estimated. The consequence assessment included illness, fluoroquinolone treatment, and persistent symptoms in patients treated with a fluoroquinolone. Using best available data to estimate the parameters and probabilities of each event, stochastic simulation was used to represent uncertainty and variability in many of the parameters. A scenario analysis was performed to evaluate the uncertainty of the following parameters: (1) probability of resistance development in treated animals, (2) portion of illnesses attributable to ground beef, and (3) probability of persistent symptoms in patients 18 years of age and over treated with a fluoroquinolone. The population at risk was restricted to people 18 years of age and over, as fluoroquinolones are not labeled for treatment of gastroenteritis in children. The mean annual increased risk of cases in the U.S. population (18 years of age and over) where compromised fluoroquinolone treatment resulted in persistent symptoms was estimated to be 1 in 61 billion (one case every 293 years) for Salmonella, 1 in 33 billion (one case every 158 years) for MDR Salmonella, and 1 in 2.8 billion (one case every 13 years) for Campylobacter.

Prevalence of Campylobacter jejuni and Campylobacter coli in market-weight turkeys on-farm and at slaughter.

To monitor the effects of feed withdrawal on the prevalence of Campylobacter, market-weight turkeys from six farms were examined before and after perimarketing events (feed withdrawal, transport, and holding at the slaughterhouse). Prior to transport, birds (n = 30 per farm) were slaughtered on-farm, and viscera (crops, duodenum, jejunum, ileum, colon, ceca, gallbladder, and spleen) were removed on the premises. Within ca. 48 h, cohorts (n = 30 per farm) from the same flock were transported to a commercial abattoir, maintained in holding sheds, slaughtered, and the viscera were removed. No differences in the prevalence of Campylobacter spp. were evident when individual flocks were compared pre- and posttransport. However, when data for the six farms were combined, Campylobacter spp. were recovered (pre- versus posttransport) at comparable rates from the duodenum (74.7 versus 74.7%), ileum (87.3 versus 92.7%), ceca (64 versus 57%), colon (86.7 versus 80%), and spleen (0 versus 0%). After feed withdrawal, transport, and holding at the abattoir, there was an overall increase in Campylobacter spp. isolated from the gallbladder at the abattoir (14.7%) when compared with on-farm levels (0%, P < 0.05). When compared with on-farm levels (3%), the overall increase in Campylobacter spp. recovered from the crops of birds at the abattoir (24%) was significant (P < 0.05), which may be associated with a detectable decline in lactic acid in the emptied crop.

Split marketing as a risk factor for Salmonella enterica infection in swine.

On-farm reduction of Salmonella carriage prevalence in pigs requires the identification of risk factors to direct interventions development. This study was designed to determine if split marketing of finishing pigs constitutes a risk factor for Salmonella infections, by comparing Salmonella prevalence in the first group of pigs selected for harvest ("first pull") versus the prevalence in the last group of pigs selected for harvest ("close out") from multiple commercial finishing lots. Nine paired samplings were conducted consisting in matched groups of pigs from individual barns as the first pull and the close out with a 4-week interval between groups. From each group, fecal and meat samples were collected, on-farm and at harvest, respectively. Fecal samples were selectively enriched, and analyzed for the presence of Salmonella, whereas meat juice samples were analyzed for the presence of antibodies against Salmonella. In 7/9 (77.8%) of the studied barns, an increase in Salmonella prevalence was observed, based on both bacteriologic and serologic analysis. Overall, there was an increase of 9.2% (p < 0.05) in bacteriologic prevalence, and 31.3% (p < 0.05) in serologic prevalence from first pull to close out groups. This study demonstrates that a significant increase in Salmonella prevalence occurs between the first and the last group of pigs harvested from finishing lots, with close out groups of market pigs posing a higher risk for Salmonella contaminations.

Species-specific alterations in Anopheles mosquito olfactory responses caused by Plasmodium infection.

Mosquitoes infected with malaria parasites have demonstrated altered behaviour that may increase the probability of parasite transmission. Here, we examine the responses of the olfactory system in Plasmodium falciparum infected Anopheles gambiae, Plasmodium berghei infected Anopheles stephensi, and P. berghei infected An. gambiae. Infected and uninfected mosquitoes showed differential responses to compounds in human odour using electroantennography coupled with gas chromatography (GC-EAG), with 16 peaks triggering responses only in malaria-infected mosquitoes (at oocyst, sporozoite or both stages). A selection of key compounds were examined with EAG, and responses showed differences in the detection thresholds of infected and uninfected mosquitoes to compounds including lactic acid, tetradecanoic acid and benzothiazole, suggesting that the changes in sensitivity may be the reason for differential attraction and biting at the oocyst and sporozoite stages. Importantly, the different cross-species comparisons showed varying sensitivities to compounds, with P. falciparum infected An. gambiae differing from P. berghei infected An. stephensi, and P. berghei infected An. gambiae more similar to the P. berghei infected An. stephensi. These differences in sensitivity may reflect long-standing evolutionary relationships between specific Plasmodium and Anopheles species combinations. This highlights the importance of examining different species interactions in depth to fully understand the impact of malaria infection on mosquito olfactory behaviour.

The malaria vector mosquito Anopheles gambiae expresses a suite of larval-specific defensin genes.

cDNAs of Anopheles gambiae Defensin 2 (AgDef2), Defensin 3 (AgDef3) and Defensin 4 (AgDef4), identified in the genome sequence, have been characterized and their expression profiles investigated. In contrast to both typical defensins and insect antimicrobial peptides generally, the newly identified defensins were not upregulated with acute-phase kinetics following immune challenge in insects or cell culture. However, mRNA abundance of AgDef2, AgDef3 and AgDef4 increased significantly during the larval stages. Promoter analysis of all three genes failed to identify putative immune response elements previously identified in other mosquito defensin genes. As previous studies failed to identify these larval-specific defensins, it seems likely that further antimicrobial peptide genes with nontypical expression profiles will be identified as more genome sequences become available.

Swine health impact on carcass contamination and human foodborne risk.

OBJECTIVE: This study measured the relationship between lesions suggestive of subclinical pig illness at harvest to carcass contamination and human foodborne risk. METHODS: Over the course of eight visits (December 2005 to January 2006), we swabbed 280 randomly selected carcasses, during normal slaughter operations, at three points in the slaughter line: skin pre-scald; the bung or pelvic cavity following removal of the distal colon and rectum; and pleural cavity, immediately before the final carcass rinse. Each swab sponge was used on five carcasses in bung and pleural cavity sampling. Swab sponges were cultured quantitatively for Campylobacter spp., Enterococcus spp., and Enterobacteriaceae spp., and qualitatively for Salmonella spp. Data on health indicators were collected for all pigs in the study (2,625 pigs) by experienced plant quality assurance personnel. RESULTS: Campylobacter spp. were recovered from the pleural cavity in 58.9% (33/56) of pools (five carcasses/pool), and in 44.6% (25/56) of pools from the bung cavity. Enterococcus spp. were recovered from 66.1% (37/56) and 35.7% (20/56) of pleural and bung pools, respectively. The most common lesion identified was the peel-out (pleuritis or adhesions), with a total of 7.1% (186/2,625 total head). Linear regression showed that for every percentage point increase in peel-outs, Enterococcus spp. contamination increased by 4.4% and Campylobacter spp. increased by 5.1% (p<0.05). CONCLUSIONS: This study showed a correlation between animal health and human health risk, as measured by carcass contamination. Therefore, animal management decisions on-farm, such as housing, antibiotic use, environment, and level of veterinary care, may directly impact public health.

Low prevalence of Listeria monocytogenes in cull sows and pork.

The goal of this study was to determine the prevalence of Listeria monocytogenes in sows slaughtered at a single Midwestern plant on two occasions (trial 1, n = 179 sows; trial 2, n = 160 sows). Fecal samples collected antemortem (trial 1) as well as animal tissues, and carcass swabs collected at the abattoir (trials 1 and 2) were analyzed. Eight isolates of L. monocytogenes were recovered from five samples that represented 0.18% of the total samples (n = 2,775). In trial 1, L. monocytogenes was detected in a tonsil sample (0.6%; 1 positive of 181 tonsils), in a carcass (0.6%; 1 positive of 179 carcasses), which was sampled prior to the organic rinse, and in two chopped meat block samples (1.2%; 2 positive of 165 samples). In trial 2, L. monocytogenes was only detected in a single chopped meat block sample (0.15%; 1 positive of 688 total samples). These data indicate the low prevalence of L. monocytogenes in the cull sow.

A stochastic assessment of the public health risks of the use of macrolide antibiotics in food animals.

Campylobacteriosis is an important food-borne illness with more than a million U.S. cases annually. Antibiotic treatment is usually not required. However, erythromycin, a macrolide antibiotic, is recommended for the treatment of severe cases. Therefore, it is considered a critically important antibiotic and given special attention as to the risk that food animal use will lead to resistant infections and compromised human treatment. To assess this risk, we used a retrospective approach; estimating the number of campylobacteriosis cases caused by specific meat consumption utilizing the preventable fraction. We then determined the number of cases with macrolide resistance Campylobacter spp. based on a linear model relating the resistance fraction to on-farm macrolide use. In this article, we considered the uncertainties in the parameter estimates, utilized a more elaborate model of resistance development and separated C. coli and C. jejuni. There are no published data for the probability of compromised treatment outcome due to macrolide resistance. Therefore, our estimates of compromised treatment outcome were based on data for fluoroquinolone-resistant infections. The conservative results show the human health risks are extremely low. For example, the predicted risk of suboptimal human treatment of infection with C. coli from swine is only 1 in 82 million; with a 95% chance it could be as high as 1 in 49 million. Risks from C. jejuni in poultry or beef are even less. Reduced antibiotic use can adversely impact animal health. These low human risks should be weighed against the alternative risks.

Risk-based analysis of the Danish pork Salmonella program: past and future.

The Danish pork Salmonella control program was initiated in 1993 in response to a prominent pork-related outbreak in Copenhagen. It involved improved efforts at slaughter hygiene (postharvest) and on-farm (preharvest) surveillance and control. After 10 years, 95 million Euros, significant reductions in seropositive herds, Salmonella positive carcasses, and pork-attributable human cases (PAHC), questions have arisen about how best to continue this program. The objective of this study was to provide some analysis and information to address these questions. The methods used include a computer simulation model constructed of a series of Excel workbooks, one for each simulated year and scenario (http://www.ifss.iastate/DanSalmRisk). Each workbook has three modules representing the key processes affecting risk: seropositive pigs leaving the farm (Production), carcass contamination after slaughter (Slaughter), and PAHC of Salmonella (Attribution). Parameter estimates are derived from an extensive farm-to-fork database collected by industry and government and managed by the Danish Zoonosis Centre (http://www.food.dtu.dk). Retrospective (1994-2003) and prospective (2004-2013) simulations were evaluated. The retrospective simulations showed that, except for the first few years (1994-1998), the on-farm program had minimal impact in reducing the number of positive carcasses and PAHC. Most of the reductions in PAHC up to 2003 were, according to this analysis, due to various improvements in abattoir processes. Prospective simulations showed that minimal reductions in human health risk (PAHC) could be achieved with on-farm programs alone. Carcass decontamination was shown as the most effective means of reducing human risk, reducing PAHC to about 10% of the simulated 2004 level.

Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein.

The small subunit of ribonucleotide reductase in Saccharomyces cerevisiae (RNR2) was induced 3- to 20-fold by a variety of DNA-damaging agents. Induction of the RNR2 transcript by at least one of these agents, methyl methanesulfonate, did not require protein synthesis. To identify sequences involved in the regulation of RNR2, we introduced deletions upstream of the transcription start site. Sequences required for induction were contained within a 200-base-pair region that could confer methyl methanesulfonate inducibility on the heterologous CYC1 promoter. This region contained a repression sequence and at least two positive activation sites. One of these activation sites bound RAP1, a protein known to associate with mating-type silencers and the upstream activation sequences of a number of genes. The behavior of deletions of the repression sequence suggests that induction of RNR2 may occur, at least in part, through relief of repression.

Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

Modeling the relationship between food animal health and human foodborne illness.

To achieve further reductions in foodborne illness levels in humans, effective pre-harvest interventions are needed. The health status of food animals that are destined to enter the human food supply chain may be an important, although often overlooked, factor in predicting the risk of human foodborne infections. The health status of food animals can potentially influence foodborne pathogen levels in three ways. First, diseased animals may shed higher levels of foodborne pathogens. Second, animals that require further handling in the processing plant to remove affected parts may lead to increased microbial contamination and cross-contamination. Finally, certain animal illnesses may lead to a higher probability of mistakes in the processing plant, such as gastrointestinal ruptures, which would lead to increased microbial contamination and cross-contamination. Consequently, interventions that reduce the incidence of food animal illnesses might also help reduce bacterial contamination on meat, thereby reducing human illness. Some of these interventions, however, might also present a risk to human health. For example, the use of antibiotics in food animals can reduce rates of animal illness but can also select for antibiotic-resistant bacteria which can threaten human treatment options. In this study, we present a mathematical model to evaluate human health risks from foodborne pathogens associated with changes in animal illness. The model is designed so that potential human health risks and benefits from interventions such as the continued use of antibiotics in animal agriculture can be evaluated simultaneously. We applied the model to a hypothetical example of Campylobacter from chicken. In general, the model suggests that very minor perturbations in microbial loads on meat products could have relatively large impacts on human health, and consequently, small improvements in food animal health might result in significant reductions in human illness.

Interactions between malaria and mosquitoes: the role of apoptosis in parasite establishment and vector response to infection.

Malaria parasites of the genus Plasmodium are transmitted from host to host by mosquitoes. Sexual reproduction occurs in the blood meal and the resultant motile zygote, the ookinete, migrates through the midgut epithelium and transforms to an oocyst under the basal lamina. After sporogony, sporozoites are released into the mosquito haemocoel and invade the salivary gland before injection when next the mosquito feeds on a host. Interactions between parasite and vector occur at all stages of the establishment and development of the parasite and some of these result in the death of parasite and host cells by apoptosis. Infection-induced programmed cell death occurs in patches of follicular epithelial cells in the ovary, resulting in follicle resorption and thus a reduction in egg production. We argue that fecundity reduction will result in a change in resource partitioning that may benefit the parasite. Apoptosis also occurs in cells of the midgut epithelium that have been invaded by the parasite and are subsequently expelled into the midgut. In addition, the parasite itself dies by a process of programmed cell death (PCD) in the lumen of the midgut before invasion has occurred. Caspase-like activity has been detected in the cytoplasm of the ookinetes, despite the absence of genes homologous to caspases in the genome of this, or any, unicellular eukaryote. The putative involvement of other cysteine proteases in ancient apoptotic pathways is discussed. Potential signal pathways for induction of apoptosis in the host and parasite are reviewed and we consider the evidence that nitric oxide may play a role in this induction. Finally, we consider the hypothesis that death of some parasites in the midgut will limit infection and thus prevent vector death before the parasites have developed into mature sporozoites.

Quantity and distribution of Salmonella recovered from three swine lairage pens.

The quantity of Salmonella recoverable from three lairage pens in a swine abattoir was determined. Using dry four-ply cotton gauze pads measuring 10 by 10 cm, 100 fecal slurry samples were collected from each of the three pens. Salmonella recovery was expressed as the log CFU per milliliter of sample. Mean values were 2.5 log CFU/ml in pen A, 2.7 log CFU/ ml in pen B, and 0.89 log CFU/ml in pen C. Median values were 2.6 log CFU/ml in pen A, 2.0 log CFU/ml in pen B, and below the detectable limit in pen C. In pen C, Salmonella was not recoverable from a high number of samples. Pen B results suggested spatial dependency, i.e., samples close together were more similar than samples farther apart. These results indicate that Salmonella concentrations vary within and between lairage pens. Because of the limited number of pens assessed, it was not possible to identify factors that were associated with the observed variation in Salmonella concentrations within and between pens. However, this variation suggests that numeroussamples are required to adequately describe the concentration of Salmonella in a lairage pen.

Effect of preslaughter events on the prevalence of Salmonella in market-weight turkeys.

The goal of this study was to determine if preslaughter events, such as transport to and holding at the slaughterhouse, affect Salmonella prevalence in turkeys. Floors of transport crates were swabbed after loading and prior to transport at the farm (time 1, n = 100 swabs per trial) and after transport to and holding at the abattoir (time 2, n = 100 swabs per trial). In addition, environmental samples were taken at each of the six premises (n = 25 per premises) as well as in the holding shed at the abattoir (n = 25 samples per trial). At slaughter, the crops, ceca, and spleens were cultured (n = 50 each per flock). As shown from the culture of the crate floor swabs collected pre- and posttransport, when individual farms were analyzed, samples from only one premises exhibited a statistically significant change, as seen by the decline in Salmonella prevalence posttransport (P < 0.01). When the data from all farms were combined, Salmonella was recovered more frequently from swabs collected pretransport at loading on-farm (time 1, 47.6%) than from swabs collected after transport (time 2, 39.7%, P < 0.01). This suggests that transport to and holding at the abattoir do not increase the prevalence of Salmonella in turkeys. This observation contrasts with the increase in Salmonella prevalence reported for hogs and some broilers.

Salmonella prevalence in market-age turkeys on-farm and at slaughter.

The objective of this study was to compare the prevalence of Salmonella in market-age turkeys on-farm and at slaughter (i.e., before and after feed withdrawal, catching, loading, transportation, and preslaughter holding). Thirty birds were randomly selected from each of 6 commercial turkey flocks scheduled to be loaded and shipped to the abattoir during the evening of the same day. Selected birds were euthanized on the farm, and the cloacal contents, large intestine, crop, ceca, liver and gallbladder, and spleen were aseptically collected. At the abattoir, 30 birds from the same flock were randomly selected from the slaughter line, and the crop, ceca, liver and gallbladder, and spleen were collected for subsequent culture at the laboratory. All flocks studied were positive for Salmonella at slaughter. No statistical difference was found between the overall prevalence on-farm and at slaughter. At both sampling points, the overall prevalence found was 33.3%. Diverging prevalence estimates were obtained based on the different sample types collected on-farm and at slaughter. In both cases, cecal content samples had the highest relative sensitivity (73.3% on-farm and 68.3% at slaughter). This study demonstrates that the preslaughter practices of feed withdrawal, catching, loading, transportation, and holding do not significantly alter the prevalence of Salmonella in market-age turkeys. Therefore, our results suggest that it may be possible to monitor the Salmonella status of turkey production farms based on samples collected at the abattoir.

Rapid aerosol transmission of Salmonella among turkeys in a simulated holding-shed environment.

Once turkeys arrive at Midwest processing plants, they are usually held in large open-sided sheds for 1 to 4 h, waiting to be unloaded. In hot, humid weather, large fans are used to cool the birds. The resultant air currents distribute a significant amount of dust to the turkeys. The dust created in this environment could be a factor in the number of Salmonella-contaminated turkeys entering slaughter plants. The objective of this study was to determine if rapid transmission of Salmonella in turkeys could occur from exposure to Salmonella-contaminated dust similar to what may be experienced in holding sheds or in other high-dust environments prior to slaughter. In the first experiment, trials of 3 different concentrations of Salmonella (1.2 x 10(9), 2.6 x 10(7), and 2.6 x 10(5) cfu/g) were conducted to determine if transmission of Salmonella enterica serovar Typhimurium var. typhimurium(chi)4232 to turkeys 2 to 4 h after aerosol exposure to contaminated feces is possible. Results showed that turkeys became infected after 2 h of exposure to airborne-contaminated feces with a concentration level of 2.6 x 10(5) cfu of Salmonella Typhimurium/g. In the second experiment, consisting of 3 trials, 1 bank (5 cages wide and 3 cages high) of turkeys (n = 15 birds per trial) was exposed to another bank of cages of S. Typhimurium-inoculated (n = 15) birds for 2 to 4 h using a fan similar to the type in processing-plant cooling sheds. Results from this experiment demonstrated that birds could be contaminated with S. Typhimurium after 2 h of exposure. Results of both studies implicate contaminated dust as a route of rapid airborne transmission of Salmonella in turkeys. Processes that generate significant dust prior to slaughter should be regarded as critical control points for Salmonella.

Evaluating the costs of mosquito resistance to malaria parasites.

Costly resistance mechanisms have been cited as an explanation for the widespread occurrence of parasitic infections, yet few studies have examined these costs in detail. A malaria-mosquito model has been used to test this concept by making a comparison of the fitness of highly susceptible lines of mosquitoes with lines that are resistant to infection. Malaria infection is known to cause a decrease in fecundity and fertility of mosquitoes; resistant mosquitoes were thus predicted to be fitter than susceptible ones. Anopheles gambiae were selected for refractoriness/resistance or for increased susceptibility to infection by Plasmodium yoelii nigeriensis. Additional lines that acted as controls for inbreeding depression were raised in parallel but not exposed to selection pressure. Selections were made in triplicate so that founder effects could be detected. Resistance mechanisms that were selected included melanotic encapsulation of parasites within 24 h postinfection and the complete disappearance of parasites from the gut. Costs of immune surveillance were assessed after an uninfected feed, and costs of immune deployment were assessed after exposure to infection and to infection and additional stresses. Mosquito survivorship was unaffected by either resistance to infection or by an increased burden of infection when compared with low levels of infection. In most cases reproductive fitness was equally affected by refractoriness or by infection. Resistant mosquitoes did not gain a fitness advantage by eliminating the parasites. Costs were consistently associated with larval production and egg hatch rate but rarely attributed to changes in blood feeding and never to changes in mosquito size. No advantages appeared to be gained by the offspring of resistant mosquitoes. Furthermore, we were unable to select for refractoriness in groups of mosquitoes in which 100% or 50% of the population were exposed to infection every generation for 22 generations. Under these selection pressures, no population had become completely refractory and only one became more resistant. Variations in fitness relative to control lines in different groups were attributed to founder effects. Our conclusion from these findings is that refractoriness to malaria is as costly as tolerance of infection.